2^nd EURO GEROSCIENCE Conference Posters

Posters

Artificial Intelligence and Aging

P01 — New Biomarkers for Alzheimer Diagnosis and Early Detection of Mild Cognitive Impairment

Aurora Román-Domínguez¹, Mar Dromant¹, Consuelo Borrás¹ (1. University Of Valencia — València (Spain))

Background: Alzheimer’s disease (AD) is the most common form of dementia and is estimated to affect more than 46 million people around the world. The most studied biomarkers for AD diagnosis are β-amyloid deposits determined by positron emission tomography (PET), glucose consumption in the brain by PET, structural magnetic resonance imaging (MRI), and cerebrospinal fluid biomarkers such as β-amyloid, tau, and phosphorylated tau. However, these biomarkers present severe problems due to their invasive nature and/or high costs. Consequently, the study of biomarkers in peripheral blood is still of great interest. In addition, a new approach to improve AD diagnosis has evolved in recent years using machine learning and other artificial intelligence (AI) systems. These models often use the mentioned blood biomarkers to predict cognitive decline and AD. In previous studies, we proposed a classification model which uses a set of biomarkers composed of 3 proteins that can be measured in serum samples. These proteins are the regulator of calcineurin 1 (RCAN1), clusterin (CLU), and receptor for advanced glycation endproducts (RAGE). Objectives: This work aimed to validate the relative contribution of blood levels of RCAN1, CLU, and RAGE to the AD diagnosis in a new cohort and to assess the relevance of the contribution of a new parameter, the malondialdehyde (MDA), as a biomarker of oxidative stress. Moreover, a secondary goal was to develop an improved predictive model for AD diagnosis with the addition of clinical parameters such as age, sex, and apoE genotype. At the same time, we studied the within-subject evolution of these blood-based parameters in a 5-year difference in the longitudinal clinical cohorts. Methods: Blood samples were obtained from the longitudinal and retrospective cohort study composed of cognitively healthy (CTL) subjects over 65 years old, some of which developed mild cognitive impairment (MCI). In addition, blood samples were also obtained from a nursing home cohort composed of dementia and AD patients. From each subject, we obtained serum and plasma samples at baseline and 5 years later, along with the clinical data of age, sex, and apoE genotype. Serum levels of CLU, RCAN1, and RAGE were measured by enzyme-linked immunosorbent assay (ELISA). Plasma levels of MDA were determined by high-performance liquid chromatography (HPLC). In a cohort-based study, we compared the levels of CLU, RCAN1, RAGE, and MDA in each longitudinal group as a case-control study. Additionally, we compared these biomolecules between the stable CTL and MCI converter longitudinal groups at baseline, when both groups were clinically diagnosed as CTL. In a study based on clinical diagnosis, we compared the 4 biomolecules between the transversal clinical groups CTL, MCI, and AD. Moreover, we developed a classification model using CLU, RAGE, RCAN1, and MDA blood levels, and age, sex, and apoE genotype as predictors to classify subjects in positive or negative for AD (AD+/AD-) by binomial logistic regression. A total of 147 observations were used to build the model. To validate these models, fold cross-validation was performed. In addition, ROC curves and other performance metrics such as accuracy, sensitivity, and specificity were determined. Results: In the cohort-based study, a decrease in CLU and RCAN1 occurred in a 5-year interval in AD and CTL subjects correspondingly. No significant changes were observed in RAGE or MDA levels in any cohort. However, when CTL and MCI healthy subjects were compared at baseline, lower levels of RAGE and higher levels of MDA were already detected. In the study based on clinical diagnosis, lower levels of MDA and RCAN1 were detected in the AD group compared to CTL and MCI. No significant differences were found in CLU or RAGE levels. Finally, we build a classification model based on the clinical diagnosis with an accuracy of 89.8 %, a sensitivity of 75.0 %, a specificity of 93.3 %, and an area under the curve (AUC) of 93.9 %. Conclusion: The study of cognitive impairment in CTL subjects that later develop MCI did not show any change in blood concentrations of CLU, RAGE, RCAN1 nor MDA in a 5-year difference. Interestingly, a difference in RAGE and MDA was already detected when comparing stable CTL and MCI converter subjects at baseline, when both groups are clinically indistinguishable. Moreover, when compared at a transversal level, no differences were found between CTL and MCI subjects, whereas AD patients were different in terms of RCAN1 and MDA. Finally, we created a classification model using blood-based parameters, along with the age, sex, and apoE genotype of the subject, which classifies a subject in AD+/AD- with an accuracy of 89.8 %.

P02 — Technological Development and Clinical Applicability of Wearable Devices for Remote Monitoring of Older Adults Health: The Senior Mobile Health Platform

Álvaro Maciel¹, Eujessika Silva¹, Daniella Carvalho¹, Paulo Barbosa² (1. Universidade Federal Do Rio Grande Do Norte — Natal (Brazil), 2. Universidade Estadual Da Paraiba — Campina Grande (Brazil))

Background: Technologies applied to health have become an increasingly present discussion among the modalities of therapeutic, diagnostic, and monitoring interventions in the health area. The health and functional capacity are a prerequisite for active elderly, which emphasizes the intermediary role of technology in quality of life. We implemented a technological strategy, based on Internet of Things (IoT) and Artificial Intelligence (AI), for health services. Developing a remote monitoring system for the elderly, which captures patient information 24 hours a day through wearable devices. Currently it is possible for Senior Mobile Health professionals to provide continuous evaluation about elderly health, offering crucial data for the prediction of adverse health conditions through smart algorithms. Therefore, the main goal of this study was to show the development of remote monitoring system and its functionally. The system consists in a web dashboard to store and view the information about the health indicators, mobile app to facility the data collect by the health professionals during the intervention and smart algorithms developed from the sensors. Methods: The Senior Mobile Health (SMH) platform has a microservice architecture, with several services connected to user and information management, data science and IoT. Most of the artificial intelligence algorithms work with time series analysis and machine learning to monitor important data on the health of the elderly. Using sensors as accelerometer, gyroscope, global position system (GPS) and the photoplethysmography sensor, we developed smart algorithms to follow the health indicators such as heart rate variability, nocturia, gait speed and others. The mobile app is designed to encourage elderly in their self-care through specific goals and activities. Results: By using wearable devices, mobile application and web dashboard, the smart algorithms of the SMH platform predict adverse health outcomes and promote real-time and continuous monitoring of the elderly health. Such algorithms are currently analyzing, for example, heart rate variability, steps, nocturia, quality of sleep and gait speed, which are closely linked to adverse outcomes in the health of the elderly. These functionalities are important in human aging process. The SMH platform receives heart rate data from the wearable devices and transform the data in information about Heart Rate Variability (HRV). The HRV reflects the oscillations in the interval between consecutive heartbeats that result mainly from the dynamic interaction between the parasympathetic and the sympathetic system. The changes in HRV patterns provide information about the decline in health, mainly the cognitive functioning. Step counting sensors, as accelerometers, can be able to daily monitor the activity level and the number of steps each day. They can be used also as a strategy to motivate the elderly to remain active through established previously goals by the health professionals. Recent studies show how many steps a day are enough for specific populations as elderly to be considered as free of chronic disease and active, or as elderly with chronic disease and sedentary lifestyle. Thereby, using the SMH platform, patients are monitored, and specific profiles are created. The nocturia is a condition in which an individual has to wake up at night many times to urination, which each urination being preceded and followed by sleep. By using sensors as accelerometers and photoplethysmography to analyze the movements and steps at night, besides the heartbeats analysis with the smart algorithms, the SMH platform can be able to identify how many times the individual wake up at night and go to the bathroom. These conditions can be associate with serious illnesses such as prostate cancer, diabetes, and others. Sleep disturbances are common symptoms among the people. However, previous studies show that elderly become more vulnerable to sleep disorders than other individuals, showing a decreasing global sleep time at night, increasing sleep latency, with sleep—wake cycle disorders, especially excessive daytime sleepiness. The monitoring of sleep patterns and sleep quality can be useful to assess the sleep influences on outcomes that interfere in the elderly health. The gait speed has been used as another important metrics to monitor. Gait speed patterns change can be markers of various general medical conditions, including, cognitive decline, and increased chance of falls, hospitalizations, or even death. The high association between gait speed and clinical adverse outcome become this measure an important information and widely used. The platform proposal is to automatically obtain this information from devices, decreasing and facilitating the work of health professionals. This platform has been implemented in several places such as public and private Brazilian hospitals to monitoring the inpatients, an Elderly Community Center to following active older adults, a Condominium for Senior to create a digital environment and other. Conclusion: This paper presented the SMH platform, a system oriented to the elderly care. The platform is registered in the Brazilian National Institute of Intellectual Property, with the patent BR512020002492-0. Results show that the platform provides interesting features of elderly monitoring.

P03 — Heart Rate Variability Remote Monitoring Using Wearable Devices

Álvaro Maciel¹, Eujessika Silva¹, Jose Marmol², Jose Bullejos², Paulo Barbosa³ (1. Universidade Federal Do Rio Grande Do Norte — Natal (Brazil), 2. Universidad De Granada — Granada (Spain), 3. Universidade Estadual Da Paraiba — Campina Grande (Brazil))

Background: The Heart Rate Variability (HRV) reflects the oscillations in the interval (ms) between consecutive heartbeats (R-R intervals) that result mainly from the dynamic interaction between the parasympathetic and the sympathetic system. The changes in HRV patterns provide an early information about the decline in health, mainly the cognitive functioning. Efficient autonomic mechanisms provide a high HRV, meaning a good adaptation for intrinsic and extrinsic factors, an indicator of healthy individuals. The HRV can be considered an early biomarker from cognitive impairment in elderly population. HR is characterized by consecutive heartbeats, being measured by counting the number of heart beats per minute (bpm). In this case, HR has a smaller spacing of 1 second. This means that this time series has the HR value every 1 second. However, wearable devices are saving data with an interval of 1-N sec, normally every 5 sec and, at this moment, it is a limitation of the manufacturer in all its devices. Therefore, the aim of this study was to show the development of a method to the remote monitoring of the HRV data of older adults, using the time series from wearable devices with photoplethysmography sensor and comparing data with those provided by a reference clinical tool, the Kubios HRV. Methods: The time series from the wearable devices are saved in the database in the shortest possible intervals. The data coming from devices has gaps (missing data) in the time series. Thus, identify these gaps and find the right method to filling the time series is a crucial step to provide the remote monitoring in HRV service. The data pre-processing is done before applying any method for the HRV analysis, since any measurement in the time series can significantly interfere in such analysis. HRV artifacts can be divided into technical and physiological artifacts. Technical artifacts include detection of missing or misaligned beats, while physiological artifacts include ectopic beats and arrhythmic events. Data acquired from devices that consists of a time series with gaps due to possible measurement failures is filled in to guarantee the integrity of the data. We chose a conventional imputation method to ensure that the data has been filled in a satisfactory way. We adopted the Mean Squared Error (MSE), which determines the average of the squared difference between the original and the predicted values, where a smaller MSE implies in a greater accuracy. In the next step, the Inter-beat Interval (IBI), also called RR-interval, is calculated using the following equation: HR = 60000/IBI. After that, we approximate the data obtained from devices to the data coming from ECG sensors, assuming that there is an unknown underlying function that consistently maps those two. Finally, the IBI data is adjusted to remove any physiological artifacts in the time series that can interfere in the analysis. Finally, the HRV analysis method is calculated. The hrv-analysis library, which is distributed under the GPLv3 license, provides different methods to remove these inconsistencies, being: Malik Rule — IBIs differing by more than 20% from the one preceding it are removed. Karlsson Rule — IBIs diverging by more than 20% of the mean of previous and next IBI are removed. Kamath Rule — This method considers a heartbeat abnormal whenever the IBI increased by more than 32.5% or decreased by more than 24.5% when compared to the previous IBI. Acar Rule — IBIs differing by more than the 20% of the mean of last nine IBIs are removed. For this study, we adopted the Kamath Rule method, since it provided the most similar results to Kubios. After that, we obtain the mean and its respective standard deviation of each IBI, in a given time interval, which through mathematical techniques unfold in some statistical indices that constitute the HRV analysis in the time-domain. Results: We compared the HRV metrics obtained by the devices with the ones calculated by Kubios. For that, we used the data collected from the 20 community-dwelling older adults and compared the executions of data analysis over the HRV metrics in this sample both using the SMH HRV algorithms and the Kubios. The results showed that our method can provide reliable results associated with HRV metrics and it can be incorporated by health professionals during the remote monitoring of their patients. The long-term recording will support the healthcare professional in assessing reactions of their patients’ autonomic nervous system during normal daily activities and in response to therapeutic interventions, by comparing the patient’s daily readings to determine if any significant changes have taken place. Conclusion: For the health professionals, the routine of usage and the acquisition of HRV metrics in the long-term provide continuous following of the older adult health and early risk identification that involve the individual during the human aging process giving for the health professionals better conditions to follow and care their patients.

P04 — A Multi-Tissue Meta-Analysis of Methylation Variability: Uncovering Robust Markers for Biological Ageing

Kirsten Seale¹, Namitha Mohandas¹, Nir Eynon¹, Sarah Voisin¹ (1. Victoria Univeristy — Melbourne (Australia))

Background: The DNA methylation landscape (‘the methylome’) accrues numerous, widespread changes over the lifespan. Of importance are two distinct phenomena: differential and variable patterns of DNA methylation. Specific DNA methylation sites (‘CpGs’) that change with chronological time, capture the DNA methylation patterns that are shared by all individuals over the lifespan. These are known as age-associated differentially methylated positions (DMPs), which have been used to build ‘epigenetic clocks’ capable of predicting chronological age with high accuracy. In contrast, a significant proportion of CpG sites exhibit increased variability with age, known as age-associated variably methylated positions (VMPs). Unlike DMPs, VMPs do not necessarily shift in their mean methylation, but instead increase in variance over time, resulting from aberrant and unpredictable changes. VMPs are therefore DNA methylation patterns that are different between individuals over the lifespan, and may better reflect aspects of ‘biological ageing.’ Biological ageing, although not well-defined, represents the tissue and organismal functional status and age-associated risk for disease. Two individuals with identical chronological ages (and patterns of DMPs) may display divergent patterns across VMPs. Focusing only on patterns of DMPs is limiting when trying to understand aspects of biological ageing, particularly when making sense of why individuals of the same age may display vastly different ageing rates. However, there is a lack of research focusing specifically on VMPs in different tissues. Moreover, whether VMPs drive meaningful changes relevant to biological ageing remains largely unknown. Objectives: The overall objective of this research is to uncover the tissue-specific and tissue-independent DNA methylation signatures of biological age. Specifically, we aim to uncover robust DNA methylation sites that diverge, or increase in variability, with age, and build a comprehensive atlas of the unique and overlapping signatures of biological ageing in human tissues. This research will address the following research questions: 1. What are the tissue-specific and tissue-independent patterns of variable DNA methylation (VMPs) in the human methylome that accrue with age? 2. Is variable DNA methylation biologically meaningful? Methods: To rigorously determine the methylation signatures that change in variability with age, we will perform a large-scale, multi-tissue Epigenome-wide Association Study (EWAS) meta-analysis of age. To accomplish this, we aim to collect the methylomes of over 40,000 human samples from ~140 datasets, across 15 tissues and cell types, profiled on the Illumina Methylation array platforms (27K, 450K and 850K). The first component in this analysis will be a tissue-specific EWAS meta-analysis of age. The Breusch-Pagan test for heteroscedasticity will be used to identify the CpGs that change in variability with age (i.e. VMPs). EWAS will be conducted in each dataset separately, adjusting for covariates (i.e. sex), and results will be pooled using a fixed-effects meta-analysis. Then, we will conduct a multi-tissue EWAS random effects meta-analysis, pooling the results from datasets across all tissue types (i.e. blood, muscle, skin, etc). For robustness, only VMPs that are present in at least 60% of the datasets will be included for downstream biological interpretation. Results: We present preliminary results from the tissue-specific meta-analysis of age in whole blood of 11,141 samples aged 14 to 94, from 11 datasets. We identified 120,866 VMPs (16.6% of tested CpGs) at FDR < 0.005, nearly all of which increase in variability with age. Importantly, this increase in variability was not driven by changes in cell type proportions with age, as results remained largely unchanged when the meta-analysis was repeated after correction for blood cell types (Pearson’s correlation of meta-analysis Z-scores = 0.92, p-value < 2.2e-16). We then investigated the distribution of VMPs in chromatin states profiled in PBMCs and found VMPs to be over-represented in bivalent enhancers and regions repressed by the Polycomb complex, and depleted in active transcription start sites (χ2 test p-value < 2.2e-16). Gene ontology (GO) pathway enrichment showed VMPs are linked to developmental and cell signalling pathways (FDR < 0.005). Conclusions: To our knowledge, our study is the largest, genome-wide investigation of DNA methylation variability and age to date. Our large sample size has facilitated the discovery of 120,866 VMPs, suggesting variable DNA methylation is pervasive throughout the ageing methylome. Although the increase in variance at each VMP is small, collectively, VMPs seem to gradually introduce noise at key regions related to development and differentiation. Our results therefore suggest biological ageing may result from a decline in cellular function, as cells lose the ability to maintain their identity with age. Quantifying the DNA methylation changes that become noisy and unpredictable with age is challenging and leans on the availability of large datasets to accurately capture sites that ‘unravel’ across the lifespan. We present encouraging preliminary results in a single tissue, which we aim to expand on in a multitude of human tissues. This research therefore holds the potential to significantly contribute to the current body of knowledge, by painting a global picture of biological ageing in the human methylome. The authors report no conflicts of interest.

P05 — Infrared Spectroscopy as a Diagnostic Tool for Osteosarcopenic Women

Ricardo Guerra¹, Raysa Freitas¹, Daniel Freitas¹, Igor Oliveira¹, Gerlane Guerra¹, Gustavo Duque², Kassio Lima¹ (1. Federal University Of Rio Grande Do Norte — Natal (Brazil), 2. University Of Melbourne — Melbourne (Australia))

Background: Osteosarcopenia is characterized by the presence of both sarcopenia and osteopenia or osteoporosis and increases prevalence of disability, falls, fractures and risk of mortality. The access to methods that could early detect these musculoskeletal disorders is unpredictable to avoid severe damages to the older population health. In face of the importance to develop strategies to prevent diseases and disabilities related to aging, there is a need to purpose faster and more effective ways to measure the impact of aging in order to reduce them. The Fourier transform infrared (FTIR) spectroscopy is a fast and reproducible technique that requires small amounts of sample and highly sensitive to the molecular structure of biological tissues components such as lipids, proteins and nucleic acids. Objectives: The aim of this study is to analyze the diagnostic power of the FTIR spectroscopy to differ community-dwelling older women with and without osteosarcopenia through serum. Methods: This is a diagnostic cross-sectional study approved by the Federal University of Rio Grande do Norte Ethics Committee in Research, under number 2.368.206. Those women who met the inclusion criteria underwent a structured interview and a body composition assessment through DXA in order to observe appendicular muscle mass and bone mineral density. Sarcopenia was diagnosed according to the revised EWGSOP criteria: handgrip strength <16kgf (low muscle strength) and Appendicular Skeletal Muscle Mass <15kg (reduced muscle mass). Osteopenia/osteoporosis when femoral neck t-score was ≤ -1, as the recommended by the International Society for Clinical Densitometry and the Brazilian Society for Clinical Densitometry. Blood samples were obtained and analyzed at the same laboratory and by the same technician. They were centrifuged per 4000rpm per 10 minutes to obtain the serum, which was kept in aliquots storage at -80°C for posterior analysis. Before spectrometric analysis, all samples were defrosted in ambient temperature for 30–40 minutes and the protein precipitation process was made. Precipitation was performed by adding 1.5µ of perchloric acid at 7M to a 100µ aliquot of serum. The aliquot was placed in a vortex (FlexVortex 2, Loccus®) per 15 seconds, and centrifuged per 12 minutes, 12.000rpm and at 4°C. Precipitate samples were analyzed thought a spectrometer SHIMADZU® model IRAffinity-1 equipped with an ATR accessory. The spectra were obtained with a resolution of 4 cm-1, 34 seconds (32 scans) per spectrum. An amount of precipitate mass was placed at the ATR crystal and a spectrum was obtained. Then, a mathematical model was created using multivariate classification techniques that denoted the graphic spectra of the molecular groups. Results: Our sample comprised thirty-two osteosarcopenic women. The same amount of non-osteosarcopenic women were included as controls. FTIR spectral data in the fingerprint region (900 — 1,800 cm-1) were pre-processed through Savistzky-Golar smoothing followed by automatic weighted least squares (AWLS) baseline correction and vector normalization in order to avoid non-biological interference that could impair the results. Training set was composed by 70% of samples (22 negative and 22 positive) and 30% of samples (10 positive and 10 negative) was used for test models. The preprocessed spectra were used to obtain several classification techniques and the most feasible model was GA-SVM, achieving 80.0% of accuracy, 70% of sensitivity and 90% of specificity. The selected wavenumbers selected by the model to differ groups were: 952, 975, 995, 1134, 1165, 1246, 1247, 1309, 1317, 1477, 1500, 1517, 1533, 1556 and 1579. These wavenumbers represent potential biomolecular structures that would possibly differ osteosarcopenic from non-osteosarcopenic women, as carbohydrates, amino acids, lipids and nuclei acids. Conclusion: Osteosarcopenia has been widely discussed in the last years and few studies were conducted with this population. Therefore, this study is unprecedented in using an infrared method to diagnosis this condition. FTIR has an overall good accuracy, sensitivity and specificity, which implies that this technique is suitable to early diagnosis osteosarcopenia in older women.

P06 — The Discovery and Characterization of a New Premature Aging Disease

Daniela Bakula¹, Morten Scheibye-Knudsen¹ (1. Center For Healthy Aging, University Of Copenhagen — Copenhagen (Denmark))

Background: The global increase in the elderly population is a considerable challenge confronting societies worldwide. Since the prevalence of most chronic diseases increases exponentially with age it is critical to improve our understanding of aging mechanisms at the cellular and organismal level. To understand the molecular basis of aging, monogenic premature aging diseases represent good models. Objectives: We aimed to identify and characterize new premature aging disorders. Methods: We used in silico, in vitro and in vivo analysis to understand the pathogenesis of a new premature aging disorder. Results: Through machine learning algorithms we identified that the disease Woodhouse-Sakati syndrome closely resembles aging phenotypes. The disease is characterized by diabetes, hearing loss, hair loss and neurodegeneration and is caused by mutation in the DCAF17 gene. We describe the first Scandinavian family suffering from Woodhouse-Sakati syndrome and show through multiomics and deep learning approaches that the patients display features of aging including increased cellular senescence. We discover that loss of DCAF17 leads to accelerated aging in mice and in cell lines, that DCAF17 is recruited to the DNA damage site and that loss of DCAF17 leads to altered DNA metabolism. We further mapped the DCAF17 interactome to identify substrates of the DDB1-DDA1-DCAF17 protein complex and discovered a single conserved arginine required for the function of this complex. Conclusion: In sum, our study revealed that the disorder Woodhouse-Sakati syndrome can be classified as a new premature aging disorder caused by defective DNA metabolism.

P07 — SCALLOP: A Novel Computational Method for the Quantification of Age-Related Increase in Transcriptional Noise at the Single-Cell Level

Olga Ibáñez-Solé¹, Alex M. Ascensión¹, Marcos J. Araúzo-Bravo¹, Ander Izeta¹ (1. Biodonostia Health Research Institute — Donostia-San Sebastián (Spain))

Background: Aging has been associated to a loss of cell type identity, presumably caused by cumulative DNA damage. This phenomenon has been referred to as an increase in cell-to-cell variability, heterogeneity or transcriptional variation/noise, and measured using different computational methods in several tissues and organisms, with conflicting results. However, it was not clear whether the divergence between studies could be attributed to the methods used for the quantification of noise or to cell type-specific or tissue-specific effects. Objectives: First, we aimed to develop and validate a novel method (Scallop) for the quantification of transcriptional noise based on the degree of membership of each individual cell to its assigned cell type cluster. Second, we aimed to use this method to determine whether aging is associated to a generalized transcriptional deregulation, by measuring whole-tissue transcriptional noise in several single-cell RNAseq (sc-RNAseq) aging datasets. Third, we aimed to study cell type-specific effects related to transcriptional noise and cell type composition in the aging lung, using published murine and human datasets. Methods: The robustness of Scallop to its input parameters was validated on five single-cell RNAseq datasets. The greater transcriptional stability of cells with a high membership to clusters was shown in a dataset of peripheral blood mononuclear cells. Sc-RNAseq datasets of different cell types and tissues (murine and human pancreas, dermal fibroblasts, brain and lung) were downloaded and preprocessed using Scanpy. Cell type annotations of the same tissue and species were harmonized. Whole-tissue transcriptional noise was quantified in the young the old fraction of each dataset using Scallop. In addition, four published methods for the quantification of noise were implemented in Python and used as a control: biological variation over technical variation, Euclidean distance to cell type mean, Euclidean distance to tissue mean on set of invariant genes and global coordination level (GCL). Transcriptional noise was measured in four murine and two human datasets using Scallop and the four alternative methods, scaled using the robust scaler from scikit-learn and the difference between old and young cells was computed. Cell type composition effects were studied by building generalized linear models (GLM) on the composition data while accounting for age-related effects. Results: We developed Scallop, a tool for transcriptional noise quantification in sc-RNAseq datasets and validated its robustness to input parameters (number of bootstrap iterations and fraction of cells used). Membership is associated to a higher transcriptional stability, as shown by a greater statistical significance obtained with stable cells in differential expression analysis. We showed there is no conserved pattern of increase in transcriptional noise associated to aging, using several aging datasets: human and murine pancreas, human and murine dermal fibroblasts, murine brain and human and murine lung. We observed that some tissues show an age-related increase in transcriptional noise, yet these changes are not conserved across different datasets of the same tissue. Also, we observed a great variability associated with the noise quantification method. A deeper analysis on the murine and human aging lung confirmed that increase in transcriptional noise is not conserved at the cell type level across datasets and organisms. However, we found important changes in cell type composition with aging, particularly affecting in the immune cell fraction. A significant enrichment of plasma cells was found in all aging lung datasets. Changes in the community of alveolar macrophages were also found, as they were significantly depleted in most murine datasets, but significantly enriched in human datasets. A deeper analysis on alveolar macrophages revealed that in humans, four distinct alveolar macrophage communities emerge with aging from a single transcriptionally homogeneous cluster. We show that the four aged alveolar macrophage subclusters show a markedly different expression of genes coding for surfactant proteins (SFTPC, SFTPA1, SFTPA2, SFTPB and SFTPD). We show that one of the main differences between Scallop and distance-to-center methods is that the former could accurately detect the emergence of distinct and transcriptionally stable alveolar macrophage subpopulations, whereas the rest of the methods would interpret this as a single population undergoing loss of identity. Conclusion: Scallop is a robust, unbiased method for the quantification of transcriptional noise and is able to discern transcriptionally stable clusters from noisy cells that cannot be robustly assigned to a cell type cluster. Aging is not universally associated to an increase in transcriptional noise, as there are important cell type-specific and tissue-specific effects. The aging lung is characterized by important changes in the cell type composition, particularly of the immune cell fraction. Plasma cells are enriched in all murine and human aging lung datasets. Alveolar macrophages suffer important changes in abundance: they are significantly depleted in murine aged lungs and significantly enriched in human aged lungs. In humans, subpopulations of alveolar macrophages with different surfactant expression profiles emerge with aging. The authors declare there are no conflicts of interest.

Cellular Senescence and Senolytics

P09 — Transcriptome Analysis Reveals Senescence Pathways Driven by EGR1, DDX11L1 and MIR454 Associated with Frailty

Ander Matheu¹, Ander Saenz-Antoñanas¹ (1. Biodonostia Institute — San Sebastian (Spain))

Frailty is a geriatric syndrome that represents a state of vulnerability with increased risk of negative health outcomes. The molecular mechanisms underlying frailty remain poorly understood. Transcriptomic analysis of robust and frail community-dwelling individuals identified the differential expression of 35 transcripts associated with frailty based on 3 different scales. Serially passaged human primary fibroblasts and myoblasts also showed a differential expression pattern of selected candidates. Among the 35 identified transcripts, additional analyses revealed the expression of a 3-gene pattern, i.e. increased EGR1 and reduced DDX11L1 and miR454, robustly associated with frail individuals and with clinical parameters related to frailty such as polypharmacy and multimorbidity. Loss and gain of EGR1, DDX11L1 and miR454 function experiments in fibroblasts showed they regulate cell proliferation and senescence. In addition, they modulate senescence-related downstream pathways. In summary, our results identify a pattern of 35 genes whose altered expression is related to frailty and highlight senescence-associated pathways driven by EGR1, DDX11L1 and miR454 as potential biomarkers and players in the physiopathology of frailty.

P10 — Ribosome Heterogeneity by Rrna Methylation in Skin Cell Senescence

Markus Schosserer¹ (1. Institute Of Molecular Biotechnology, Department Of Biotechnology, University Of Natural Resources And Life Sciences, Vienna — Vienna (Austria))

Background: The accumulation of senescent cells is associated with several age-related pathologies and represents a primary driver of skin aging. Senescence cells cause tissue damage by secreting a mix of pro-inflammatory cytokines and extracellular matrix remodeling factors. Objectives: This study aims to elucidate how ribosomes and protein translation change in cellular senescence. Particularly we focus on alterations of the methylation patterns of ribosomal RNA. Methods: We exposed human dermal fibroblasts to doxorubicin to induce cellular senescence and tested if specific methylations of ribosomal RNA contribute to the senescent phenotype. We also measured total protein synthesis by OPP incorporation and specific translation of senescence-associated mRNAs by polysome profiling. Results: Global protein synthesis, ribosome biogenesis, and nucleolar size surprisingly increased in senescent compared to contact-inhibited quiescent cells. Furthermore, several differentially methylated sites, either hyper- or hypo-modified, were present in the rRNA of senescent and quiescent compared to proliferating cells. Methylation levels showed a correlation with the expression of snoRNAs installing these modifications. By inhibiting specific snoRNAs, we confirmed the causality of these modifications for either promoting or inhibiting cell proliferation. Conclusion: Our combined data suggest that even subtle modifications of the ribosomal RNA might have profound and precise effects on cellular physiology and contribute to the heterogeneity of ribosomes. Ribosomal RNA modifications and translation might provide future targets for the selective elimination of senescent cells or blocking their harmful secretome.

P11 — Sevs from Young Adscs Improve Healthspan and Prevent Frailty in Old Mice

Jorge Sanz-Ros¹, Cristina Mas-Bargues¹, Aurora Roman-Dominguez¹, Mar Dromant¹, Nekane Romero-Garcia¹, Jose Viña¹, Consuelo Borras¹ (1. University Of Valencia — Valencia (Spain))

Background: In societies where older persons are a growing demographic group, it is a public health priority to add health to the years lived, as well as to promote the ability to live independently. Frailty is the state before disability, not only preventable but also treatable. Its early diagnosis allows interventions that delay it, preventing disability and all the consequences that it entails, both on a personal, family, social and economic level. Therefore, the search for strategies to delay the degenerative changes associated with aging and frailty is especially interesting. The results of most investigations show that the mesenchymal stem cell-conditioned environment or its components mediate some of its biological functions. Small extracellular vesicles (sEVs) are membrane vesicles containing proteins, miRNAs, lipids, and metabolites that could serve as a platform for the development of new therapies. Compared to cells, sEVs are more stable, do not present a risk of aneuploidy, have a lower possibility of immunological rejection after allogeneic administration in vivo, and can therefore provide a safer therapy for various diseases. Objectives: To study the effect of treating old mice with sEVs derived from young ADSCs on several tissues affected by aging and frailty-related parameters. Methods: We isolated ADSCs from the inguinal fat pads of young mice (4–6 months), we then isolated sEVs from the culture media of ADSCs using differential ultracentrifugation, for the characterization of sEVs we used flow cytometry and electronic microscopy with gold labeling of CD63. All animals used were C57BL/6J of both sexes, physical tests in mice were assessed by the same person blindly. Urea was used as a marker of renal function and was determined with a commercial kit. Oxidative stress was studied using a protein oxidation kit and MDA determination by HPLC. Senescence in tissues was studied with the loss of the LMNB1 marker in histological slices. Metabolites were determined using Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy. miRNAs studies were carried in an Affymetrix miRNA 4.0 array. Ratios comparing physical test and plasma values after treatment compared to baseline were determined and plotted as % over baseline. The baseline was defined as 0%. All groups were tested for the presence of outliers with the ROUT method (Q=2%). Saphiro-Wilk test was conducted in each comparison to test the normality of each group. Unpaired Student’s t-test or Mann-Whitney test were used to calculate the p-value for pairwise comparisons. For the in vitro experiments, ANOVA was used with Tukey’s multiple comparisons as posthoc test. GraphPad Prism 9.0 software was used for the analysis. Results: We injected old animals (20–24 months) in the tail vein with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals and found an improvement of several functional parameters usually altered with aging, such as motor coordination, grip strength, fatigue resistance, fur regeneration, and renal function. Frailty index analysis showed that 40% of old control mice were frail whereas none of old ADSCs-sEVs treated mice were frail. This functional improvement was accompanied by molecular and structural benefits in muscle and kidney. ADSCs-sEVs induced pro-regenerative effects, as well as a decrease in oxidative stress, inflammation, and senescence markers. The metabolome of mice treated with ADSCs-sEVs changed to a youth-like pattern. Finally, we gained some insight into the miRNAs contained in sEVs that might be, at least in part, responsible for the effects observed. Conclusions: We propose that treatment with sEVs from young MSCs modulate important processes associated with aging and therefore can be useful against frailty promoting healthy aging. We state that we do not have any conflict of interests.

P12 — Exploring the Role of Arginine-Rich Peptides and the Nucleolus in Ageing and Neurodegeneration

Oleksandra Sirozh¹, Vanesa Lafarga¹, Oscar Fernández-Capetillo¹ (1. Cnio Spanish National Cancer Research Centre — Madrid (Spain))

Ageing is a physiological organism-wide decline in homeostasis and function, accompanied by a number of consolidated molecular hallmarks. Nucleolar alterations are emerging as a novel hallmark of ageing, however, a causative link remains elusive. Notably, genetic and pharmacological inhibition of both ribosome biogenesis and translation extends lifespan in a number of model organisms, strongly linking decreased nucleolar function to longevity. Argininerich dipeptide repeats (DPRs) accumulate at nucleoli, cause nucleolar stress, and lead to cell death. Importantly, these DPRs are implicated in neurodegeneration, namely C9ORF72 amyotrophic lateral sclerosis (ALS) and spinocerebral ataxia 36 (SCA36). Through an in vitro approach, we have previously shown that a generalised DPR coating of DNA and RNA leads to protein displacement from nucleic acids, and results in cell death. Such displacement of ribosomal and nucleolar proteins induces nucleolar stress. To test this in vivo, we have developed a novel mouse model based on tissue-wide inducible expression of DPRs. Unexpectedly, the animals develop a lethal premature ageing phenotype within 3 months, suggesting the implication of nucleolar stress in ageing. Surprisingly, DPR toxicity is ameliorated by the mTOR inhibitor rapamycin, or by downregulation of MYC, both of which are known to extend lifespan in mammals. We propose that a generalised inhibition of the cell’s protein metabolism reduces DPR-mediated toxicity and nucleolar stress, and extends lifespan in these animals. Our data and ideas about this project will be discussed.

P13 — Relationship Between Sarcopenia and Water Cellular Profile in Community-Dwelling Older People: Results from PRO-EVA Study

Álvaro Maciel¹, Sabrina Fernandes¹, Rafaella Gonçalves¹, Dimitri Guedes¹, Saionara Camara¹, Philipe Barreto² (1. Universidade Federal Do Rio Grande Do Norte — Natal (Brazil), 2. Gérontopôle De Toulouse — Toulouse (France))

Background: Sarcopenia is considered a muscle disorder characterized by a progressive reduction in muscle quantity/quality associated with a decline in strength. This condition leads to a series of physical limitations, reduction in activities of daily living, in functionality, which can lead to fractures, institutionalization and death. Thus, it is essential to assess muscle mass, which can be performed through bioelectrical impedance analysis (BIA). This method is widely used in epidemiological studies because it is easy to access, simple, non-invasive and inexpensive. BIA measures the differences in resistance of different tissues and, therefore, becomes useful to assess the water composition, in addition to measurements that allow the analysis of the individual’s cellular health conditions. During the aging process it is common for a reduction in total body water levels and cellular dehydration to occur. Some studies have reported that these factors can impair muscle function, causing catabolism, hypotrophy and, consequently, predisposing to the development of sarcopenia and frailty. In parallel to this, the phase angle, also measured by BIA has been seen as an influencing factor in muscle components and in the development of sarcopenia. This measure is considered a good parameter for cellular health and integrity. It is known that water indices and phase angle may be related to sarcopenia, but there are no studies in the literature that have evaluated other measures of water and cellular health in elderly people with and without sarcopenia. Objectives: Describe the water and cellular health profile in community-dwelling older people with and without sarcopenia. Methods: This is a cross-sectional study composed of 702 community-dwelling older people living in Parnamirim, localized in Rio Grande do Norte (RN), aged over 60 years and of both genders. The present study is a secondary analysis of the study data that proposes the implementation a global geriatric assessment system based on a digital platform for decision making, monitoring and promoting active aging (PRO-EVA study) — approval numbers: 2,996,329. Sociodemographic, anthropometric and body composition data (muscle mass index, phase angle, capacitance, resistance, reactance, body cell mass, extracellular mass, basal metabolic rate, total body water, intracellular water and extracellular water) were collected through the BIA analysis. Sarcopenia was defined based on diagnostic criteria and the cutoff point proposed by the European Working Group on Sarcopenia in Older People 2 (EWGSOP2). For data analysis, the Statistical Package for Social Science (SPSS) 20.0 was used. To compare the “non-sarcopenic” and “sarcopenic” groups in relation to each variable included in the present study, Student’s t test and Chi-square test were performed. In addition, the effect size in each comparison was calculated using Cohen’s d. Results: A total of 702 community-dwelling older people were evaluated, of which 7.5% were diagnosed with sarcopenia. It can be observed that sarcopenic older people have a higher mean age (73.3±8.1; p-value 0.002). Regarding body composition variables, it is noted that sarcopenic older people have a lower mean BMI, calf circumference, and skeletal muscle mass index. In addition to having worse results for grip strength and gait speed, when compared to non-sarcopenic older people. Regarding the BIA variables according to the EWGSOP2, it can be analyzed that sarcopenic older people have lower means for all analyzed variables, when compared to those who do not have sarcopenia. We can highlight as average differences between the body capacitance (155,03 and 95% CI: 115.3 — 194.7); resistance (-3.99 and CI: -105.9 — (-71.3)); body cell mass (kg) (4.92 and 95% CI: 4.04 — 5.81); extracellular mass (kg) (4.41 an 95%CI: 3.47 — 5.48); basal metabolic rate (190.04 and 95%CI: 236.3 — 343.7); intracellular water (kg) (3,68 and 95%CI: 3.02 — 4.35) and extracellular water (kg) (2.63 and 95%CI: 2.01 — 3.25). As for the effect size, the variables have a large effect size, except for the phase angle (0.38), total body water (0.54) and reactance (0.23). Conclusion: With the analyzed results, we concluded that the presence of sarcopenia can lead to a reduction of the variables related to the water and cellular health profile in the older people. In addition, it is worth noting that the aging process itself leads to a slow and gradual process of dehydration and high osmotic stress, which can compromise the function and structure of the protein responsible for muscle contraction, causing serious defects in the muscles, catabolism, anabolic resistance and consequently, loss of muscle mass, which influences the onset of sarcopenia. Therefore, it is important to carry out longitudinal studies so that it is possible to understand the real influence of sarcopenia on the cellular health of community-dwelling older people.

P14 — Endothelial-Derived Circulatory MIR34 Reinforces Senescence in Progeria

Selma Osmanagic-Myers¹, Christina Manakanatas², Santhosh Kumar Ghadge², Roland Foisner², Viviane Fleischhacker¹, Eleonora Nardini^1, (1. Institute Of Medical Chemistry, Center For Pathobiochemistry And Genetics, Medical University Of Vienna — Vienna (Austria), 2. Max Perutz Labs, Center For Medical Biochemistry, Medical University Of Vienna, Vienna Biocenter Campus (vbc) Vienna (Austria))

Aging is a significant risk factor for development of cardiovascular diseases (CVDs) and this is of crucial importance in view of the fact that global life expectancy is increasing. Using endothelial specific “Hutchinson-Gilford progeria syndrome (HGPS)” disease mouse model (Prog-Tg) we could recently show that age-related endothelial dysfunction has strong systemic effects causing cardiac fibrosis and development of diastolic dysfunction. Here we analyzed the molecular signaling pathways that cause endothelial dysfunction in HGPS and nature of paracrine “systemic” effects with an aim to ameliorate deleterious systemic changes. Using coculture model systems and analysis of non-endothelial cell populations from the lung tissues of Prog-Tg mice we could demonstrate dramatic cell non-autonomous (paracrine) effects of HGPS endothelial cells on non-endothelial populations leading to accumulation of immune cell infiltrates in the vicinity of blood vessels but also prominent general paracrine senescence in the neighboring tissue. mRNA and micro RNA (miR) transcriptome analysis using NGS sequencing followed by GO enrichment analysis from freshly isolated primary endothelial cell extracts including miR sequencing in plasma revealed upregulation of p53-linked senescence and senescence-associated secretory pathway (SASP) together with deregulation of many miRs. Among these, significantly upregulated senescence-associated miR34 and miR31 were also found upregulated in plasma suggesting systemic tumor suppressive effects. Consistent with this, both miRs were found upregulated in lung tissue, bone marrow but only miR34 was also found upregulated in cardiac tissue and aortic arch regions suggesting the key enrolment of this miR in cardiovascular pathology of Prog-Tg mice. On the mechanistic level miR34 knockdown ameliorates the p53-linked senescence phenotype with concomitant upregulation of Sirt1 but also reduces the levels of late stage senescence marker cyclin-dependent kinase inhibitor 2a, p16INK4. These findings indicate that miR34 keeps the senescence phenotype in “checkmate” position by acting on two separate senescence regulating branches p53-and p16-axis with potentially systemic tumor suppressive effects particularly in cardiovascular tissue. Altogether, assessment and targeting of miR34 might offer novel options for prognosis and treatment of cardiovascular pathology in HGPS but also geriatric and chemotherapy treated patients with abundant endothelial senescence phenotype.

P15 — Senescent Cells: Role in Countering Pulmonary Hypertension Development and Progression

Emmanuelle Born¹, Marielle Breau², Larissa Lipskaia¹, Amal Houssaini¹, Delphine Beaulieu¹, Elisabeth Marcos¹, Rémi Pierre³, Marcio Do Cruzeiro³, Geneviève Dérumeaux¹, Dmitry Bulavin⁴, Jesùs Gil⁵, David Bernard⁶, Jean-Michel Flamand⁶, Shariq Abid¹, Serge Adnot¹ (1. Inserm U955 — Créteil (France), 2. Crcm — Marseille (France), 3. Inserm U1016 — Paris (France), 4. CNRS — Nice (France), 5. Mrc — London (United Kingdom), 6. Umr Inserm U1052/CNRS 5286 — Lyon (France))

Background: Senescent cells (SCs) are involved in proliferative disorders but their role in the pathogenesis of pulmonary hypertension (PH) remains undefined. We investigated cell senescence in patients with pulmonary arterial hypertension (PAH) and explored the role played by SCs in animal models of PH by using suicide gene or senolytic strategies and by disabling the p16 pathway. Methods: Investigation of senescence (p16, p21) and DNA damage (γ-H2AX, 53BP1) markers in patients with PAH and in murine models; monitoring of p16 activation by luminescence imaging using p16-luciferase (p16LUC/+) knock-in mice; induction of SC clearance using p16-ATTAC mice carrying a p16 promoter-driven killer gene construct or the senolytic drugs ABT-263 or FOXO4-DR peptide; inactivation of p16 using p16LUC/LUC mice. PH was investigated in mice exposed to normoxia, chronic hypoxia, or hypoxia plus Sugen, in mice overexpressing the serotonin transporter (SM22-5-HTT+) and in rats given monocrotaline. Results: Patients with PAH compared to controls exhibited high lung p16, p21, and γ-H2AX proteins levels, with abundant vascular cells co-stained for p16, γ-H2AX and 53BP1. Hypoxia increased thoracic bioluminescence in p16LUC/+ mice and lung levels of senescence and DNA damage markers in WT mice together with senescence-associated secretory phenotype (SASP) components and strong p16 staining of pulmonary endothelial (PECs) and smooth muscle cells (PASMCs), PECs representing 30% of lung SCs during normoxia. Elimination of SCs in p16-ATTAC mice or in WT mice treated with ABT263 increased right ventricular systolic pressure, the RV hypertrophy index, and vessel remodeling with increased dividing PCNA-stained vascular cells during both normoxia and hypoxia, together with marked reduction in lung PECs. Altered pulmonary hemodynamics and reduced lung PECs were also observed in aged p16LUC/LUC mice, in WT mice subjected to Sugen or hypoxia+Sugen, and in SM22-5-HTT+ mice treated with either ABT263 or FOXO4-DRI peptide compared to their respective controls. Monocrotaline-induced PH in rats was slightly reduced by one-week ABT-263 treatment but aggravated at 3 weeks with reduction in lung vascular ECs. Conclusion: SCs protect against PH development and progression. Elimination of senescent PEC by senolytic interventions may underly pulmonary hemodynamics deterioration. Disclosures: The authors have no conflicts of interest to disclose.

P16 — Glyoxal Induces Senescence in Primary Human Keratinocytes Through Oxidative Stress and Activation of the AKT/FOXO3A/P27KIP1 Signaling Pathway

Rym Halkoum¹, Virginie Salnot², Christophe Capallere³, Christelle Plaza³, Aurore L’honoré⁴, Karl Pays⁵, Bertrand Friguet⁴, Carine Nizard⁵, Isabelle Petropoulos⁴ (1. Sorbonne Université (biological Adaptation And Ageing, B2a-Ibps) And Lvmh Recherche (life Science Department) — Paris (France), 2. 3p5 Proteom’ic Facility, Université De Paris, Institut Cochin, Inserm, Cnrs — Paris (France), 3.Ashland, Global Skin Research Center, Advanced Skin Research & Bioengineering Dept — Sophia Antipolis (France), 4.Sorbonne Université, Cnrs, Inserm, Institut De Biologie Paris-Seine, Biological Adaptation And Ageing, B2a-Ibps — Paris (France), 5. Lvmh Recherche. Life Science Department — Saint-Jean-De-Braye (France))

Background: Senescence is a well-characterized cellular response to telomere shortening or stress exposures that provoke DNA damages accumulation and/or oxidative stress. This process is associated with specific markers such as permanent cell proliferation arrest, an increased senescence-associated β-galactosidase (SA-βGal) activity and the secretion of messenger molecules by cells expressing the Senescence-Associated Secretory Phenotype (SASP). The SASP, which composition depends on many factors such as the cell type or the nature of the stress that induces senescence, contributes to the reinforcement and propagation of the senescent phenotype and aging through its autocrine and paracrine roles. Since the skin constitutes a barrier with the external environment, it is particularly subjected to different types of stresses, and consequently prone to premature cellular aging. Glyoxal, a dicarbonyl compound formed during glycation reaction, glucose autooxidation or lipid peroxidation, is a precursor of Advanced Glycation End-products (AGEs), whose presence marks normal and pathological aging. Objectives: We wanted to know if glyoxal could induce normal human epidermal keratinocytes (NHEK) senescence and, if so, to determine which molecular events were involved in the establishment of the senescent phenotype. Moreover, we wanted to distinguish the signaling pathways involved during the first steps of the glyoxal-induced senescence process (early-stage senescence) and those activated once the senescent phenotype is fully established (late-stage senescence). Also, we were interested in the characterization of the resulting secretory phenotype during the early-stage senescence induced by GO (or GASP for Glyoxal-Associated Secretory Phenotype). By studying the molecular mechanisms and secretory phenotype involved in the “early-stage” senescence, we wanted to bring new insights regarding anti-aging strategies by acting on the “early-stage” paracrine effect, thus preventing the installation and propagation of the senescent phenotype in the organism. Methods: Proliferative NHEK cells were submitted to repeated and subcytotoxic doses of 200 µM or 300 µM of GO for two days. Experiments were then conducted at day 3 for the early-stage senescence and at day 8 for the late-stage senescence characterization. Gene expression and protein level of senescence markers were assessed by qRT-PCR and Western Blot respectively. Since cell cycle arrest is one of the main features of the senescence, we investigated the effect of GO treatment on NHEK proliferation using EdU incorporation. Reactive Oxygen Species and AGEs levels were determined using CellRox probe and Western Blot. To study the early-stage GASP, we used different methods based on qRT-PCR, Luminex assay and mass spectrometry. Finally, we evaluated the consequences of GO exposure ex vivo on reconstructed human epidermis by studying the morphological alterations using histological staining and the senescence markers expressions using qRT-PCR and immunofluorescence. Results: We showed that exposure of proliferative NHEK to low-range doses of GO induces oxidative stress and protein glycation leading to premature senescence. Such senescence bears a unique molecular progression profile and occurs in two phases : the early phase is marked by cell cycle arrest and decreased LMNB1 gene expression and the late phase manifests the positive SA-βGal phenotype. Analysis of the signaling pathways underlying NHEK cell cycle arrest during the early-stage led to the identification of the AKT/FOXO3a/p27KIP1 pathway. Following GO exposure, AKT is dephosphorylated leading to FOXO3a nuclear translocation where it activates the expression of the cell cycle marker, p27KIP1. Surprisingly, in the epidermis, the activation of this pathway is restricted to GO-induced senescence, since no activation of p27KIP1 was observed in NHEK cells submitted to replicative senescence. We demonstrated that maintenance of the cell cycle arrest required for the establishment of the late-stage senescence phenotype is accompanied by activation of the p16INK4/pRb pathway while the AKT/FOXO3a/p27KIP1 pathway activation is no longer. In addition to the upregulation of a large number of well-known “core SASP” factors, characterization of the secretome conducted to the identification of 152 proteins whose secretion was specific to GO exposure. Among these, 4 proteins may represent new biomarkers of early GASP. Finally, ex vivo experiments conducted on reconstructed human epidermis showed that GO treatments result in the epidermal thinning that correlates to increased expression of p16INK4 but not p27KIP1, indicating that GO treatment leads to a late-stage phenotype of the reconstructed epidermis 9 days after exposure. Conclusion: Our results bring new insights regarding the molecular mechanisms implicated in senescence and the characterization of the secretory phenotype in human keratinocytes, which represents a less studied cellular model compared to fibroblasts. Hence, senotherapeutics aimed at modulating the GASP holds promising potential by acting on its paracrine effect, thus preventing the propagation of the senescent phenotype and delaying skin aging.

P17 — Using the P21-Mtert Bypassing Senescence Model to Study the Role of Telomeres in Age-Related Osteoarthritis

Christina Fissoun¹, Margot Milano¹, Laura Braud², Marielle Breau², Yves-Marie Pers¹, Christian Jorgensen¹, Vincent Geli², Jean-Marc Brondello¹ (1. Irmb Umr1183 — Montpellier (France), 2. Crcm U1168 — Marseille (France))

Background: Osteoarthritis (OA) is the most common degenerative disease of the joint which correlates with articular senescent cell accumulation. Studies on articular chondrocytes isolated from OA patients reveal telomeres shortening and significant induction in cell cycle inhibitors including p21Cdkn1a and p16INK4a that drive senescence onset. Our hypothesis is that dysfunctional premature telomeres in the joint could be at the root of the DNA damage driven senescence onset during OA development. Objectives: using a knock-in mouse model in which telomerase reverse transcriptase (TERT) is expressed from the p21Cdkn1a promotor that can be activated in response to telomere dysfunction in order to prevent spontaneous age-promoting OA. Methods: Comparative bone and joint analysis between 18 months-old p21+/+, p21-/+ and p21+/mTERT mice using micro-computed tomography (micro-CT), confocal laser scanning microscopy (CLSM) and immuno-histochemistry-staining for senescence marker expression. Results: We found a significant decrease in cartilage thickness, hallmark for OA phenotype, in p21+/mTERT aged mice compared to the same age p21+/+ and in p21-/+ mice. In contrast, we could reveal that articular sub-chondral bone from p21+/mTERT mice exhibit a significant lower thickness and volume with p21+/- but not p21+/+ genotypes. Additionally, the tibial cortical bone volume in these mice is also reduced compared to p21+/- but not p21+/+ revealing a premature osteoporosis. Conclusion: Altogether, the expression of the catalytic form of telomerase under p21 promoter favors OA development in old mice. Collectively, our data propose for the first time a differential role for the telomerase and p21 in bone and cartilage compartments to maintain joint homeostasis during lifespan.

P19 — E4F1-Mediated Control of Pyruvate Dehydrogenase is Essential for P53-Dependent Senescence

Pierre-François Roux¹, Jean-Marc Lemaitre², Matthieu Lacroix¹, Claude Sardet¹, Laurent Le Cam¹ (1. Ircm, Institut De Recherche En Cancérologie De Montpellier, Inserm U1194, Université De Montpellier, Institut Régional Du Cancer De Montpellier — Montpellier (France), 2. Irmb, Institute For Regenerative Medicine And Biotherapy, Inserm U1183, Chru Montpellier Saint-Eloi Hospital — Montpellier (France))

Background: Cellular senescence is a response to numerous non-lethal stress types that result in a persistent and stable cell cycle arrest. Senescence-inducing stimuli include progressive telomere shortening but also many other stimuli that do not impact telomere integrity such as abnormal oncogenic signaling, DNA damage, oxidative stress or enhanced cytokine signaling. Numerous studies in rodents and primates, including humans, support the notion that the progressive increase in the number of senescent cells in various organs impacts on tissue homeostasis during aging. Moreover, genetic or pharmacological elimination of senescent cells in mouse models delays the onset of age-related pathologies and extends median and maximum lifespan, indicating that cellular senescence directly contributes to physiological aging. Senescence is a multistep biological process during which cells experience profound modifications of chromatin architecture associated with epigenetics and gene expression changes. However, the mechanisms underlying these modifications remain largely unknown. Moreover, converging evidence indicates that senescent cells also undergo a metabolic reprogramming, but its molecular consequences, in particular on the epigenome, have been so far poorly investigated. At the molecular level, cellular senescence involves the activation of two well-characterized molecular cascades implicating the Retinoblastoma (pRB) and the p53 tumor suppressors that play pivotal functions in this process. p53 is a master transcriptional regulator of genes controlling cell proliferation, senescence, DNA repair and cell death. Interestingly, its tumor suppressive activities have been more recently extended to the control of cellular metabolism. However, the mechanism(s) by which p53 controls its target genes implicated in metabolism, and its role in physiological responses to metabolic challenges are not fully understood. In addition, the importance of p53-mediated control of cellular metabolism during cellular senescence and aging remains to be determined. The multifunctional protein E4F1 regulates a complex transcriptional program involved in metabolism through both p53 -dependent and -independent mechanisms. Initially identified as a cellular target of the viral oncoprotein E1A during adenoviral infection, E4F1 was found to interact with several components of key oncogenic pathways including pRB, p53, BMI1 and p14ARF. Using whole-genome chromatin immunoprecipitation approaches combined with next generation sequencing (ChIP-seq), our lab unraveled an important function for E4F1 in the control of the pyruvate dehydrogenase (PDH) complex (PDC), a multimeric metabolic enzyme localized in mitochondria that catalyzes pyruvate oxidation into Acetyl CoEnzyme A (AcCoA). Objectives: The exact roles of the metabolic rewiring occurring during senescence remain to be further explored but previous data indicate that some of them impinge on the induction of the pro-inflammatory phenotype of senescent fibroblasts. Nevertheless, more work is necessary to evaluate how metabolic reprogramming directly contributes to the initiation or maintenance of cellular senescence and to identify the regulators of these metabolic changes. Methods: Here, using gain and loss of function approaches, we demonstrate that the pro-senescence activities of E4F1 relate to its ability to control the PDC and to control p53 functions at early steps of cellular senescence. Results: Expression of ectopic E4F1 in primary fibroblasts induced premature senescence that relied on a functional Cdkn2a-p53 pathway, whereas E4F1 deficiency protected against Ras-induced senescence. The pro-senescence property of E4F1 associated with the induction of pyruvate dehydrogenase (PDH) activity at early steps of senescence and genetic inhibition of PDH was sufficient to bypass premature senescence induced by ectopic E4F1. Increased PDH activity preceded the induction of a p53 response that resulted in a profound metabolic reprogramming associated with changes in lipid metabolism, increased mitochondrial biogenesis and fusion, and enhanced mitochondrial-ROS production which ultimately led to cellular senescence. Conclusion: Altogether, our results show that E4F1 functions in pyruvate metabolism and its ability to control p53 synergistically contribute to the early steps of cellular senescence.

P20 — Senolytics Rejuvenate the Reparative Activity of Human Cardiomyocytes and Endothelial Cells

Piotr Sunderland¹, Lulwah Alshammari¹, Emily Ambrose¹, Georgina Ellison-Hughes¹ (1. Centre For Human And Applied Physiological Sciences & Centre For Stem Cells And Regenerative Medicine, School Of Basic And Medical Biosciences, Faculty Of Life Sciences & Medicine, King’s College London — London (United Kingdom))

Background: Senescent cells have emerged as bona fide drivers of ageing and age-related cardiovascular disease, with senescent cells accumulating in the aged heart and following damage/injury. Removal of senescent cells using senolytics can prevent or delay tissue dysfunction, physical dysfunction and extend health- and lifespan. In the heart, we have shown that senolytics can rejuvenate the regenerative capacity of the aged heart. Objectives: Here, we investigate the effects of cell senescence on human cardiomyocyte and endothelial cell viability, survival, proliferation and senescence in vitro. We also determine the actions of the senolytics, Dasatinib (D) and Quercetin (Q), on the viability, survival, proliferation and angiogenesis of human cardiomyocytes and endothelial cells in vitro. Methods: To this aim, we have developed a transwell insert co-culture stress-induced premature senescence model system in vitro to investigate the direct and selective effects of human cell senescence and senescent human cell clearance by D+Q. In this co-culture system, human iPSC-cardiomyocytes (iPSC-CMs) or HUVECs were seeded on the top chamber insert, and DOX-induced, senescent cardiac progenitor cells (CPCs) or senescent HUVECs were seeded on the bottom chamber. Cultures were left for 7 days and then cardiomyocytes or HUVECs in the top chamber were analysed for viability, proliferation and markers of senescence, p16INK4A, SA-β-gal, and γH2AX, and the conditioned medium analysed for SASP factors. The cultures were then treated with D+Q (0.5µM D with 20µM Q) for 3 days to clear the senescent cells in the bottom chamber. and then 7 days later the iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and the markers of senescence, p16INK4A, SA-β-gal, and γH2AX, and conditioned medium analysed for SASP factors (total of 17 days). Results: Co-culture of senescent CPCs with iPSC-CMs leads to decreased iPSC-CM viability and proliferation. There is no increase in senescence in iPSC-CMs. Treatment with senolytics D+Q resulted in rescue of viability but not proliferation. Similar effects were observed with HUVECs where non-senescent HUVECs showed decreased viability and proliferation when co-cultured with senescent HUVECs. These results were confirmed by treating non-senescent HUVECs with conditioned media collected from senescent cells, which showed decreased viability, proliferation and tube formation of HUVECs. D+Q conditioned media treatment ameliorated HUVEC viability, proliferation and tube formation. Luminex analysis of the senescent cell conditioned media revealed upregulation of SASP factors, but the level of SASP factors were reduced with application of D+Q. Conclusion: In this work we show a crosstalk between human senescent cells and non-senescent cardiomyocytes or endothelial cells co-cultured in a transwell system and demonstrate that D+Q senolytics have therapeutic potential in rejuvenating the reparative activity of cardiomyocytes and endothelial cells. These results open the path to further studies on using senolytic therapy in age-related cardiac deterioration and rejuvenation

LP01 — Identifying Genes Involved in Geroconversion

Marimar Benitez¹, Andrew Koff¹, Caroline Gleason¹, Juliana Delgado¹ (1. Memorial Sloan Kettering Cancer Center — New York (United States))

Background: Aging is associated with changes in dynamic physiological processes and is a major risk factor for many diseases, including cancer. Recently, it was shown that accumulated senescent cells in aged tissue can trigger excessive inflammation, contributing to the loss of function in multiple tissues as mammals age. How senescent cells arise and why they accumulate with age is hotly debated, but there is consensus that endogenous and exogenous stresses are main contributors. While we know the mechanism by which many stressors induce cell cycle exit, and how inflammation-inducing programs can be activated, we know little about the developmental pathway, and the genetic underpinning of it, by which cells convert from a reversible growth arrested state into a stable-arrested inflammation provoking state. Objective: This work aims to understand the transition from quiescence into senescence, known as geroconversion, by identifying regulators and investigating how these genes are necessary for irreversible arrest and the inflammation-provoking senescence-associated secretory program (SASP). Methods: The Koff laboratory has focused on CDK4/6 inhibitors (CDK4/6i) therapy-induced senescence in cancer cell lines as a model of geroconversion because it can separate the decision to become senescent from reversible cell cycle exit. Using a synchronized system developed in our lab, we performed RNA sequencing at different time points along the geroconversion pathway and identified approximately 150 non-SASP transcripts whose expression might be involved in regulation of geroconversion. Many of these transcripts localize to the nucleus, where they act as transcription factors or chromatin remodelers, while others localize to the mitochondria, the lysosome, the cytosol, or the plasma membrane. In this work, we performed a transcriptomics-candidate driven screen to identify genes essential for geroconversion. Results: Our data suggest that cells treated with CDK4/6i for 7 and 14 days are preparing to become irreversibly arrested at day 7, but are still transforming their SASP transcriptional program. Also, that early prosenescent markers are induced at day 7. Furthermore, our data shows that serum-starved cells are in a quiescent state and do not express a SASP transcriptional program. Additionally, our preliminary data suggests that transcripts that encode cytosolic and secreted proteins have different profiles in quiescent vs senescent cells. However, transcripts that encode nuclear proteins, transcription factors and enzyme remodelers seem to be downregulated in both the quiescent and senescent state. Conclusion: To gain insight into the geroconversion process, this study takes advantage of a previously generated dataset to examine how cells commit to irreversible arrest in several different contexts. The results from this work will elucidate the pathways that cells take as they move from reversible to irreversible arrest, thereby identifying potential therapeutic targets to block geroconversion. Given that senescence plays a deleterious role in many organ systems during aging, precluding the accumulation of senescent cells is an attractive tactic to prevent many diseases with one approach. Finally, the results of these studies will contribute significantly to our current understanding of geroconversion, thus informing future efforts to regulate senescence. Mapping the landscape of cellular senescence under physiological or pathological conditions will undoubtedly aid the targeted development of therapeutic approaches for senescent cells.

LP02 — Towards A Better Characterization of the Role of Intrinsic (I.E. Atrogin) and Extrinsic (I.E. Senescent Cells) Factors in the Onset and Progression of Sarcopenia in Zebrafish

Aurelie Quillien¹ , Elena Morin¹, Romain Madelaine², Cedric Dray¹, Jean-Philippe Pradère¹ (1. Institut Restore, Umr 1301, Institut National De La Santé Et De La Recherche Médicale (inserm),-Cnrs-Université Paul Sabatier, Université De Toulouse, Toulouse, France. — Toulouse (France), 2. Davis Center For Regenerative Biology And Medicine, Mdi Biological Laboratory, Bar Harbor, Me 04609, Usa. — Bar Harbor (United States))

Background: One of the major challenges of our society is the aging of the population. The proportion and number of elderly people is constantly increasing, with an estimated of 2.1 billion elderly people by 2050, i.e. one person in five (WHO). However, longer life expectancy is not always associated with well-being, hence the need to study «healthy» aging. Aging is characterized by a progressive loss of physiological integrity, leading to an alteration of functions and an increase in comorbidities. This leads to the appearance of age-dependent pathologies, such as the predominantly geriatric condition sarcopenia. Sarcopenia is defined by a progressive loss of muscle mass associated with alterations in physical function and muscle quality. Multifactorial causes are described, such as defective neuromuscular function, decline in anabolic hormones, mitochondrial dysfunction, systemic inflammation, or proteostasis imbalance. Both intrinsic and extrinsic factors are involved during sarcopenia, but it is still unclear to what extent they contribute to the development of this musculoskeletal disorder and how they influence each other. On one hand we are interested by two E3 ubiquitin ligases, atrogin-1 and MuRF1, which are considered as intrinsic factors contributing to sarcopenia. In particular, these proteins promote the degradation of structural and contractile proteins in muscle cells. Interestingly, it has been shown that both genes expression is increased during aging and that their loss of function protects cells from proteolysis. They thus appear as good candidates as biosensors of sarcopenia but also as a means to induce muscle decline in an intrinsic way. On the other hand, we are also taking a closer look to cellular senescence. Recent data, suggests that both satellite cells senescence and immunosenescence are contributing to sarcopenia. For instance, in the zebrafish telomerase mutant, which has shorter telomeres and dies prematurely compare to wild type fish, an increase of senescence markers is observed in various tissues including the skeletal muscles. Objectives: Simply, our aim consist in gaining more insight on how both intrinsic and extrinsic (respectively atrogin and senescence in this study) impact separately and/or in concert the onset and the progression of sarcopenia. Methods: To better understand the etiology of sarcopenia, we are using the zebrafish as a genetic model. The zebrafish is a vertebrate whose genome is 70% conserved with humans, as well as its muscle physiology and telomere length. In order to observe and promote both sarcopenia onset and progression, we have established or are in the process of generating different transgenic lines. In the first reporter lines created, the fluorescent protein GFP is express under the control of atrogin promoters, allowing the detection of presumed sarcopenic muscle fibers. We are also generating different reporter lines to detect senescent cells. Finally, we have also produced a transgenic fish line that allows the overexpression of the atrogin1 gene in a time and muscle controlled manner via the CRE-LOX system to intrinsically promote sarcopenia. And we are also taking advantage of accelerated aging fish, the zebrafish mutant for telomerase (tert -/-). By combining, the different transgenic fish we aim to detect when and which fibers are sarcopenic, which genetic manipulation promote sarcopenia and how senescent cells are contributing to muscle aging. Results: First we have analyzed GFP expression under the control of the different atrogin promotors. Interestingly, we observed that atrogin1 drives GFP expresson principally into slow fibers while Murf1 conducts GFP expression majoritairly into fast fibers. This result suggests fiber specificity for atrogin1 and MurF. In the context of accelerated aging (tert-/-), the expression of the transgene Tg(murf:GFP) is strongly increased in the mutant fish compared to its expression in a wild type fish of the same age. Moreover, its expression is no longer restricted fast fibers but it is also present at a relatively high level into slow fibers. Conclusion: Our first data indicate that our strategy should help us gaining a better insight in sarcopenia etiology, i.e the role of both intrinsic factor and senescent cells. In long term, we plane to combine our transgenic tools with interventional approaches to decipher which regimen or pharmaceutical treatment could slow sarcopenia onset and or progression.

LP03 — Intrinsic Capacity Assessment in the Caniage Pilot Cohort

Tiphaine Blanchard¹, Géraldine Jourdan², Sophie Dupuis-Coronas², Petra Pouch-Buck³, Oceane Richard³, Giovanni Mogicato⁴, Louis Casteilla², Isabelle Raymond Letron², Nathalie Priymenko⁵ (1. Chuvac, Université De Toulouse, Envt, — Toulouse (France), 2. Restore Research Center, University Of Toulouse, Inserm 1301, Cnrs 5070, Efs, Envt — Toulouse (France), 3. Chuvac, Université De Toulouse, Envt — Toulouse (France), 4. Tonic, Toulouse Neuroimaging Center, Université De Toulouse, Inserm, Ups — Toulouse (France), 5. Umr 1331, toxalim, Inrae — Toulouse (France))

Background: Aging is the main risk factor for the development of age-related diseases as well as for the decline in intrinsic capacity (IC: cognition, locomotion, sensory, vitality, psychological). The maintenance of stable and optimal IC defines a robust individual and an healthy aging, while the decline in IC characterize a state of frailty that lead to dependence. In this context, research studies on aging and so on “Geroscience” become a new challenge for scientific community. The pet dog represents a particularly interesting animal model of spontaneous aging. The dog project, “CaniAge cohort”, is a long-term biological study of household dogs, that will allow to identify pertinent predictive biomarkers of healthy/pathologic aging. Companion dogs are powerful spontaneous models for studying aging because they are closer to human physiology and way of life than laboratory models. Selective breeding has generated lineages with a very wide range of sizes and associated lifespans, as well as breed-based disease risks. They are followed by a sophisticated health care system and considered as sentinels of environmental factors since they share their living space with their owners. Objectives: The main goal of the “CaniAge pilot cohort” project is built a collection of data and biological samples linked to longitudinal functional capacity assessment in order to stratify the enrolled subjects as robust, frail or disable. This new cohort participate to the INSPIRE project. INSPIRE initiative aims to build a program for Geroscience and healthy aging research going from animal models to humans. So, the “CaniAge pilot cohort” complete the already existing mice and killifish animal cohorts and represents a unique opportunity to study aging in a species close to humans, but with a much shorter lifespan than people that allows to collect results over only a few years. In order, to put in parallel the “CaniAge cohort” and the human translational cohort (INSPIRE-T cohort), the objective of this pilot cohort of 80 Dogs followed over 3 years, is to implement simple methods to evaluate the dog’s IC phenotyping in a clinical setting, using the human tests as references. Indeed, both sociodemographic data and biobanking can easily be transposed from human to pets by an owner questionnaire and similar biological sampling (blood, urines, feces, tears, swabs, and hair bulbs). In contrast, functional assessment of Intrinsic Capacities (IC) as defined by the WHO for human have not been validated for the housedog. Results: The five domains of IC of dogs in the CaniAge pilot cohort are assessed during the yearly follow-up visit as follows: — dogs’ cognition: with the Cognitive Dysfunction Syndrome Evaluation tool, a questionnaire completed by the owners about disorientation, social interactions, sleep-wake cycles, house soiling, learning, memory, activity and anxiety (DISHAA) of their dog, completed with a simple memory test (memorize the presence of a single object hidden under one of three boxes). — locomotion: several mobility tests have already been experienced on dogs, using the human chair rise test as a reference. The most significant and feasible test is the 6-min walk, measuring the distance the dog can travel in six minutes and the stair trial measuring the time to climb the stair (corrected with dog size). — vitality involves the same approaches as for the human cohort. Unintentional weight loss and loss of appetite is noticed by the owner and dogs are regularly weighted. Fatness and musculature can easily be assessed in dogs by palpation using validated scales (Body Condition Score and Muscular Condition Score, respectively) and with focused ultrasonography measures. — sensory domain is documented with a questionnaire filled in by the owner and a clinical assessment during the visit. Vision is assessed by the blinking at threat and the cotton ball tests. Hearing being generally tested on a watchful dog by hand clapping, another test is developed with the creation of a specific soundtrack comprising different frequencies and intensities to which dogs will react by Prayer reflex. — psychosocial state is assessed thanks to a questionnaire filled in by the owner about their dog’s life including social interactions and motivation to play or go for a walk. Conclusion: The presented procedure of IC evaluation in dogs allows to validate a set of simple clinical tests that will precise the functional IC phenotypes of dogs enrolled in the CaniAge cohort in a similar way as in the translational INSPIRE human cohort. This information will contribute to explore and select new pertinent biomarkers for healthy/pathologic aging in human and in dogs and thus to develop veterinary clinical trials to test promising geroprotectors.

LP04 — Deficient Chaperone-Mediated Autophagy Contributes to Joint Damage in Osteoarthritis

Irene Lorenzo-Gomez¹, Uxia Nogueira-Recalde¹, Christian García-Dominguez¹, Natividad Oreiro², J A Pinto-Tasende², Martin Lotz³, F J Blanco^1,2, Beatriz Carames¹ (1. Biomedical Research Institute Of A Coruña (inibic), Unidad De Biología Del Cartílago — A Coruña (Spain), 2. Complejo Hospitalario Universitario A Coruña, Grupo De Reumatología Clínica — A Coruña (Spain), 3. Scripps Research — La Jolla (United States))

Background: In Osteoarthritis (OA), defects in macroautophagy are evident and precede joint damage. Indeed, pharmacological activation of macroautophagy protects against joint damage and disease. Objectives: Therefore, identifying hallmarks associated with specific autophagy subtypes could shed light to fundamental mechanisms of joint disease and facilitate the development of therapeutic strategies to prevent OA progression. Methods: A gene expression analysis of 35 autophagy genes was performed from blood from a Prospective OA Cohort of A Coruña (PROCOAC) of non-OA (Age:61,44±1,16 years; BMI:25,25±0,52; Females, n=18) and knee OA subjects (Age:65,50±1,05 years; BMI:29,55±0,67; Females, n=18, OA grade III–IV) by using SYBR green array. The differential expression of candidate genes in blood (n=30/group) and knee cartilage (Non-OA, n=12; Knee OA donors, n=21) was confirmed by using Taqman Technology.HSP90AA1, a chaperone mediated autophagy (CMA) mediator, was evaluated in human knee joint tissues (i.e. cartilage, meniscus, ACL and synovium) with different KL grades (0, 2 and 4, n=3/each KL grade) and in both spontaneous aging mice (2, 6, 12, 18, and 30 months old, n=3/each time) and surgically-induced OA mice (10 weeks after surgery, n=4/each) by immunohistochemistry. The functional consequences of HSP90AA1 deficiency on inflammation, oxidative stress, senescence and apoptosis were studied in human OA chondrocytes by gene and protein expression and flow cytometry. The potential contribution of CMA to chondrocyte homeostasis was studied by assessing the capacity of CMA to restore proteostasis upon macroautophagy deficiency by ATG5 knockdown. To study the therapeutic potential of targeting CMA, HSP90AA1 was overexpressed in human OA chondrocytes. Results: 16 autophagy-related genes were significantly downregulated in knee OA subjects (p<0.05). Macroautophagy-related genes ATG16L2, ATG12, ATG4B and MAP1LC3B, were significantly downregulated (p<0.05). Interestingly, HSP90AA1 and HSPA8, CMA mediators involved in stress response and protein folding, were significantly downregulated (p<0.001). Confirmatory studies showed a downregulation of MAP1LC3B and HSP90AA1 in blood (p<0.001) and cartilage (p<0.05) from knee OA subjects. Moreover, HSP90A was reduced in human joint tissues (i.e. cartilage, meniscus, ACL, p<0.05) and associated to OA severity. In mice, HSP90A reduction was observed not only in OA (p<0.05) but also in aging (p<0.01). LAMP2A, a key CMA mediator, was also reduced in human OA cartilage and associated with aging in mice (p<0.05). Remarkably, HSP90AA1 deficiency was functionally linked to inflammation, oxidative stress, senescence and apoptosis (p<0.05). Moreover, LAMP2A activity was decreased upon HSP90AA1 deficiency, while mTOR signaling pathway, p62 and active caspase 3 were increased (p<0.05), indicating a failure in the autophagy flux that may lead to impaired lysosomal degradation and apoptosis. Human OA chondrocytes with impaired macroautophagy through ATG5 knockdown show reduced LC3II expression and induced activation of prbs6, a direct target of mTOR, p16 and p21 (p<0.05). Interestingly, HSP90A was increased, suggesting a compensatory activation of CMA in response to specific macroautophagy defects. Remarkably, HSP90AA1 overexpression itself is sufficient to protect against joint damage by decreasing mTOR signaling and senescence in human OA chondrocytes. Conclusion: Taking together, we identified HSP90A, a marker of CMA, as a key regulator of chondrocyte homeostasis underlying a relevant mechanism in OA. A better definition of the cross-talk between CMA and macroautophagy defects might reveal its role as a hallmark of OA.

LP05 — Dasatinib and Quercetin Treatment Aleviates Cognitive Impairment in Aged but Not Young Wistar Rats

Adam Krzystyniak¹, Malgorzata Wesierska², Gregory Petrazzo³, Gadecka Agnieszka³, Magdalena Dudkowska³, Anna Bielak-Zmijewska³, Grazyna Mosieniak⁴, Izabela Figiel⁵, Jakub Wlodarczyk⁵, Ewa Sikora³ (1. Laboratory Of Molecular Bases Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland) — Warsaw (Poland), 2. Laboratory Of Neuropsychology, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland), 3. Laboratory Of Molecular Bases Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland), 4. Laboratory Of Molecular Bases Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland) — Warsaw (Poland) — Warsaw (Poland), 5. Laboratory Of Cell Biophysics, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland))

Background: Cognitive decline and accumulation of senescent cells are hallmarks of aging. Senescent cells could be targeted by senolytics such as dasatinib and quercetin (D+Q). Senolytics have been shown to ameliorate symptoms of aging-related disorders in mouse models, however mechanisms by which senolytics mediate those improvements have not been fully elucidated particularly in species other than mice. Objectives: In the present study, we tried to evaluate the impact of D+Q treatment on cognitive decline in male Wistar rats and investigate weather improved cognition following D+Q treatment in aged animals is associated with changes in the peripheral level of inflammation, brain synaptic plasticity and regulation of gene expression involved in modulation of synaptic plasticity. Methods: To verify our hypothesis, we have used a method that allows complex analysis of different aspects of cognition, namely spatial learning and memory, in the active allothetic place avoidance task (AAPAT). In the AAPAT test, formation of spatial representation requires a fully functional hippocampus [26]. Results: Our studies revealed that 8 week-long treatment with D+Q decreased peripheral inflammation measured by the levels of serum inflammatory mediators (including SASP factors) in aged rats which coincided with alleviation of learning deficits and memory impairments observed in aged animals. We also observed changes in the dendritic spine morphology of the apical dendritic tree from the hippocampal CA1 neurons upon D+Q treatment. Significant improvements in cognitive abilities observed in aged rats after treatment with D+Q were also associated with changes in the trimethylation level of histone H3 at lysine 9 and 27 isolated from the hippocampus. The beneficial effects of D+Q on learning and memory in aged rats were long-lasting and persisted at least 6 weeks after the cessation of the drugs administration. Conclusion: Our results provide new insights into the mechanisms of improvement of cognitive abilities by senolytics and offer valuable data confirming the effectiveness of D+Q treatment for age-associated learning and memory deficits in rats. This work was supported by the by the National Science Center grants: UMO-2017/26/E/NZ4/00637 (JW, AK), UMO-2019/35/B/NZ4/01920 (ES, AK, MW, ES, JW, MD, GM, AB-Z, IF, GP).

LP06 — Nuclear Changes in Prematurely and Replicatively Senescent Vascular Smooth Muscle Cells

Agnieszka Gadecka¹, Marta Koblowska^2,3, Helena Kossowska², Natalia Nowak⁴, Anna Bielak-Zmijewska¹ (1. Laboratory Of Molecular Bases Of Ageing, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warszawa (Poland), 2. Institute Of Biochemistry And Biophysics, Polish Academy Of Sciences — Warszawa (Poland), 3. Laboratory of Systems Biology, Faculty of Biology, University of Warsaw — Warszawa (Poland), 4. Laboratory Of Imaging Tissue Structure And Function, Neurobiology Center, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warszawa (Poland))

Background: Ageing is inextricably linked with cellular senescence. It is believed that excessive accumulation of senescent cells is a cause of the health deterioration during aging. Cellular senescence is characterized by a permanent cell cycle arrest that can result from the natural shortening of telomeres during finite process of cell division — called replicative senescence (RS), or caused by stress agent (DNA damage, oxidative stress, etc.) — referred as premature senescence (PS). Senescent cells undergo a number of morphological and physiological changes, including significant changes at the nuclear level. The usually compact chromatin decondenses into loosened form of euchromatin, which is accompanied by general decrease or reshuffling of histone post-translational modifications (PTMs). This translates into a different gene expression profile. Objectives: The aim of the project was to examine both nuclear and epigenetic differences between PS and RS in human vascular smooth muscle cells (VSMC) and analyze their distinct gene expression profiles, which may aid the search for novel senescence markers. Methods: In the study, besides replicatively senescent cells, we applied two types of premature senescence: cells treated with 100nM doxorubicin (DOX) causing DNA damage, and 7.5 µM curcumin (CUR), which mode of action is DNA-damage independent. The general chromatin structure of DAPI stained nuclei were analyzed by CellProfiler and statistically summarized by SAS software. To determine the level of selected proteins and epigenetic marks we used both Western blot and immunocytochemistry method. Immunofluorescence analyzed by fluorescent or confocal microscopy was also used in investigating proteins’ localization and co-localization. The transcriptome profiles were obtained with microarrays, and selected genes were confirmed with qPCR. ChIP-seq analysis was applied in order to trace the enrichment profiles of two histone PTMs H3K4me3 and H3K9me3. Results: Western blot and immunofluorescence revealed a drastic loss of nearly all tested histone H3 PTMs and nuclear proteins in RS, significantly distinguishing it from PS. Moreover, the chromatin of RS assumes the most relaxed form as compared to PS and control cells. These result in different H3 PTMs enrichment pattern throughout the genome as well as distinct transcriptome profiles of RS and PS. Genes expressed only by PS cells encode proteins mostly involved in cellular migration or focal adhesion sites, while genes exclusive for RS encode cell cycle regulating proteins. Conclusion: The study reveals significant differences between PS and RS in terms of chromatin structure, protein and gene expression profiles and epigenetic patterns. This suggest, that further investigation of chromatin architecture may serve as a promising tool in recognizing the source of cellular senescence, therefore help in reducing the risk of age-related diseases, for instance atherosclerosis. This study was supported by National Science Centre grant, 2016/21/B/NZ3/00370.

LP07 — Failure to Repair Endogenous DNA Damage in SS-Cells Causes Adult Onset Diabetes

Matt Yousefzadeh¹, Ana Huerta Guevara², Paul Robbins¹, Janine Kruit², Laura Niedernhofer¹ (1. University Of Minnesota — Minneapolis (United States), 2. University Medical Center Groningen — Gronigen (Netherlands))

Objectives: One of the greatest risk factors for type 2 diabetes mellitus (T2DM) is aging, however the reason for this is not apparent. Stochastic damage accumulated over time can lead to the age-related decline in tissue homeostasis. We seek to understand if endogenous DNA damage in ß-cells is sufficient to drive loss of ß-cell function and promote the onset of diabetes. Methods: ERCC1-XPF, a structure-specific endonuclease, participates in multiple DNA repair pathways and diminished expression of ERCC1-XPF causes enhanced accumulation of endogenous DNA damage. In this study, we analyzed the impact ofasked if loss of ERCC1-XPF in the ß-cells of mice (Ins2-cre;Ercc1fl/-) was sufficient to impair glucose homeostasiscontrol, ß-cell function and promote the development of diabetes. Results: Ins2-cre;Ercc1fl/-mice which specifically delete Ercc1 in their pancreatic ß-cells showed no abnormalities in glucose homeostasis into adulthood. However, increased fed and fasting blood glucose levels and impaired glucose tolerancewas observed in Ins2-cre;Ercc1fl/- mice by 3-months-of-age. Deletion of Ercc1 reduced ß-cell mass and ß-cell function in adult Ins2-cre;Ercc1fl/- mice. Interestingly, only male Ins2-cre;Ercc1fl/-mice became obese on a standard diet, and had reduced lean mass and grip strength. However, both male and female Ins2-cre;Ercc1fl/- mice exhibited signs of insulin resistance, cellular senescence and inflammation in peripheral tissues. Conclusions: These findings demonstrate that ß-cell dysfunction and features of T2DM can be driven by endogenous DNA damage. This work provides a mechanism by which age can increase risk of T2DM. Lastly, it demonstrates that ß-cell dysfunction can act as a primary instigator of many of the features of T2DM. Conflict of Interest: LJN and PDR are co-founders of NRTK Biosciences, a start-up biotechnology company developing senolytic drugs.

LP09 — Senolytic Mixture of Dasatinib and Quercetin Differentially Impacts the Chromatin Structure in Young and Senescent Vascular Smooth Muscle Cells

Edyta Bulanda^1,2, Agnieszka Gadecka¹, Natalia Nowak³, Ewa Sikora¹, Anna Bielak-Zmijewska⁴ (1. Laboratory of Molecular Bases of Ageing, Nencki Institute of Experimental Biology — Warsaw (Poland), 2. Laboratory of Host-Microbe Interactions, Nencki Institute of Experimental Biology, — Warsaw (Poland), 3. Laboratory of Imaging Tissue Structure and Function, Neurobiology Center, Nencki Institute of Experimental Biology, Polish Academy of Sciences — Warsaw (Poland), 4. Nencki Institute Of Experimental Biology, Pas — Warsaw (Poland))

Background: Backgrounds: Experimental data have shown so far that removal of senescent cells from the organism alleviates the symptoms of aging and aging-related diseases in animal models, and a lot of effort is put into seeking safe and efficient factors suitable for human therapy. One of the promising mixtures for tissue and organ rejuvenation is a combination of dasatinib and quercetin (D+Q). Convincing data show their effectiveness, but the molecular mechanism and off target effects are not fully recognized. Our recent results revealed that treatment with D+Q improved the cognitive function of old but not young rats (Krzystyniak A. et al., 2022). We have observed characteristic changes in methylation of hippocampal histone H3, which suggests that the D+Q can influence chromatin architecture. Objectives: One of the most characteristic senescence-associated changes concerns nuclear structure and function. In senescent cells chromatin compaction is altered and the euchromatin form dominates over heterochromatin. Therefore, it raises the question whether senolytic treatment alters chromatin structure. In this study, we aimed to reveal the impact of D+Q used in non-toxic concentration on chromatin structure of senescent and non-senescent cells. Methods: To reveal the impact of D+Q on chromatin compaction, we first treated normal human cells with D+Q combination in which different dasatinib and constant quercetin concentration was used. To this end, we have chosen human vascular smooth muscle cells (VSMCs), fibroblasts and preadipocytes, and selected dasatinib concentration, which in the combination with quercetin, practically did not affect cell proliferation. To elucidate the impact of D+Q on chromatin structure during replicative senescence, we used VSMCs derived from several male donors. Cells were treated with D+Q mixture (100 nM and 5 μM, respectively) for 48 hours. Two different protocols of treatment were used. In the first protocol, cells were treated on early, middle and late passages and analyses were performed after the last treatment. In the second protocol, cells were treated only once in the late passages. The analyses of common senescent markers were performed together with analysis of chromatin structure by using CellProfiler software and detection of markers of the status of chromatin relaxation. Results: We observed that VSMCs were less sensitive to D+Q treatment than fibroblasts and preadipocytes as the established treatment only slightly impaired cell proliferation of these cells. Our preliminary data have shown that the D+Q mixture affects chromatin compaction differently in young and senescent VSMCs. Surprisingly, we observed that after treatment the chromatin structure of young cells resembles that typical for senescent ones. In contrast, the chromatin of senescent cells treated by D+Q copied the chromatin structure of young cells. Interestingly, early treatment of cells with D+Q (after the first two treatments in the triple treatment variant), despite influencing chromatin structure, did not influence their senescence. Conclusion: Our results suggest that D+Q differently affect chromatin structure in young and senescent cells resulting in an opposite response. However, chromatin alterations caused by senolytics in cells on early passages seem to be transient, as it was revealed by cell proliferation capacity and other cell senescence markers. The open question is how D+Q treatment leading to senescent cells elimination will affect chromatin structure of young cells. References: Krzystyniak A, Wesierska M, Petrazzo G, Gadecka A, Dudkowska M, Bielak-Zmijewska A, Mosieniak G, Figiel I, Wlodarczyk J, Sikora E (2022). Combination of dasatinib and quercetin improves cognitive abilities in aged male Wistar rats, alleviates inflammation and changes hippocampal synaptic plasticity and histone H3 methylation profile. Aging (Albany NY) 18;14. This study was supported by National Science Centre grant, 2016/21/B/NZ3/00370 and the Nencki Institute statutory funds.

LP10 — Aging of Neuromuscular Junctions: Alterations in Connections with the Microenvironment

Sandra Fuertes-Alvarez¹, Noelia Pelaez-Poblet¹, Ander Izeta^1,2 (1. Biodonostia, Tissue Engineering Group — San Sebastian (Spain), 2. Tecnun-University of Navarra, School of Engineering, Department of Biomedical Engineering and Science — San Sebastian (Spain))

Background: The neuromuscular junction (NMJ) is the synaptic interface through which motor neurons innervate muscle fibers. The function of NMJs is essential for muscle contraction. NMJs are formed by four elements: presynaptic nerve terminals; postsynaptic endplates organized in characteristic pretzel-like structures; terminal Schwann Cells (tSCs), which cap the endplates and Kranocytes that cover NMJs over tSCs. Both, tSCs (neural crest derived cells) and Kranocytes (perisynaptic fibroblasts) are essential for the correct function and maintenance of NMJs, since they are involved in reinnervation processes after nerve injury and the elimination of tSCs give rise to denervation of muscle fibers. Recently, we found that in homeostatic conditions tSCs and Kranocytes establish contacts with the capillaries near NMJs. Moreover, it has been described that the NMJ microenvironment (resident macrophages and vascularization) participates in the reinnervation process of NMJs after denervation. During aging, the impairment of neuro-muscular synaptic activity gives rise to muscle atrophy and declined muscle mass and function (sarcopenia). Pretzel-like structures become degraded and terminal nerves disconnect from muscle fibers. However, little is known about the tSCs and kranocytes behavior during age-related degeneration of NMJs and in relation with their connections with the NMJ microenvironment. Objective: The aim of this work is the analysis of the age-related degeneration of the structure of NMJ-capping cells, and their relation with the NMJ microenvironment during aging. Methods: Skeletal muscles from hindlimbs of C57BL/6 mice (N=3) were analyzed using fluorescence microscopy and confocal microscopy. Immunofluorescence staining was performed by whole mount. Young (up to 2 months), adult (eight to ten months) and old (twenty to twenty-four months) mice were used. Results: We observed cellular alterations during NMJ aging process, and also in their connections with NMJ-microenvironment. These data unveil new information about the NMJ-aging process and malfunction of aged NMJs underlying sarcopenia. Conclusion: These results shed light about the age-related degeneration of the tSCs and Kranocytes and their connections with NMJs microenvironment during the aging process, specifically with the vascularization and NMJ-microenvironment. Key words: Aging, neuromuscular junction (NMJ), terminal Schwann Cells (tSCs), Kranocytes, vascularization, microenvironment.

Geroscience of COVID

P21 — SARS-COV-2 Infection Causes Massive Lung-Cell Senescence

Larissa Lipskaia¹, Emmanuelle Born¹, Pauline Maisonnasse², Fouillade Charles³, Pascal Quantin², Elisabeth Marcos¹, Arturo Londono-Vallejo³, Roger Le Grand³, David Bernard⁴, Serge Adnot⁵ (1. Université Paris Est Creteil — Créteil (France), 2. Cea — Fontenay-Aux-Roses (France), 3. Institut Curie — Paris (France), 4. Université De Lyon — Lyon (France), 5. Aphp Hopital Henri Mondor — Creteil (France))

Introduction: Older age is an important risk factor for severe COVID-19 disease. Understanding the biological mechanisms that link aging to the pathogenesis of COVID-19 is essential for developing of therapeutic strategies. We hypothesized that cell senescence, a basic aging process that plays a pivotal role in lung diseases, is involved in the pathogenesis of COVID-19 including the development of long-lasting lung alterations. Methods: To evaluate the impact of SARS-CoV-2 infection on cell senescence, we 1) analyzed publicly available datasets of scRNA-seq performed in BALF cells from patients with moderate or severe/critical COVID-19; 2) investigated lung samples from cynomolgus macaques infected with 106 pfu of a SARS-CoV-2 clinical isolate. Two macaques were sacrificed at 4 days post-infection (dpi.) and two others at 30 dpi. Results: In BALF obtained within 10 days after symptom onset, the expression of several senescence markers, i.e., CDKN2A, CDKN1A (encoding p21), uPAR, CXCL8, IGFBP3, and GDF15 was significantly increased in epithelial cells in BALF from patients with severe COVID-19, suggesting that lung-cell senescence induction was contemporary of viral detection. Next, we investigated macaques at 4 and 30 dpi, corresponding respectively to the viral load peak and to the absence of detectable viral RNA in BALF (1). Immunohistochemical analysis revealed numerous SARS-CoV-2 antigen-stained cells, also co-stained for senescence markers p16- and p21. The lungs at 30 dpi no longer contained the consolidated parenchymal areas seen at 4 dpi but showed extensive lung parenchyma remodelling, with thickening of the alveoli and pulmonary vessel walls and abundant extracellular matrix deposits as assessed by collagen staining. These lesions were accompanied with massive accumulation of p16- and p21-positive cells, mostly pneumocytes II and ECs. Of note, p16 staining of most ECs was seen in pulmonary vessels, notably those occluded by thrombosis and showing intraluminal vWF staining. Cells stained for p16 were also stained for the DNA damage markers γ-H2AX protein and p53-binding protein 1. Conclusions: Our data constitute the first evidence of temporal and topographic relations between senescent-cell accumulation and pulmonary lesions induced by SARS-CoV-2 infection. Maisonnasse P, Guedj J, Contreras V, Behillil S, Solas C, Marlin R et al. Hydroxychloroquine use against SARS-CoV-2 infection in nonhuman primates. Nature 2020; 585: 584–587

Inflammation and Immunosenescence

P23 — Numtogenesis Affecting Chromosome Segregation: Insights for a Novel Approach to Understand Immunosenescence in Mice

Mónica González-Sánchez¹, Victor García-Martínez¹, Sara Bravo¹, Hikaru Kobayashi¹, Irene Martinez De Toda¹, Blanca González-Bermúdez¹, Gustavo R Plaza², Mónica De La Fuente¹ (1. Complutense University Of Madrid — Madrid (Spain), 2. Polytechnic University Of Madrid — Madrid (Spain))

Background: Reactive oxygen species (ROS) generated at the mitochondria produce mtDNA fragments that are integrated into the nuclear genome in a time-dependent way. This phenomenon, called numtogenesis, in reference to the name given to mtDNA sequences in the nuclear genome (NUMTs: NUclear MiTochondrial DNA) is the basis of the genomic instability associated with aging. NUMTS have been mostly described in the vicinity of centromeres, nevertheless, the effect that these insertions might have on chromosome segregation and cell division is yet to be clarified. With age, the function of immune cells declines, a process that is known as immunosenescence, and the capacity of T-lymphocytes to proliferate in response to an antigenic challenge exhibits a clear decrease. Although it was proposed that oxidative stress is the basis of this immune deterioration, the underlying mechanism responsible for this defective proliferative capacity is not known. Nevertheless, an age-related increase of the oxidative stress status has been described in immune cells from humans and mice, with very significant decreases of antioxidant defenses such as glutathione reductase activity and increases of oxidant compounds such as oxidized glutathione and products of lipid oxidative damage such as malondialdehyde concentrations. Objective: The aim of this study is to provide insights into the causative effect of mitochondrial DNA insertions at the pericentromeric regions that, eventually affecting proper chromosome segregation, might be responsible for the reduced lymphoproliferation characteristic of immunosenescence. Moreover, if these mitochondrial DNA insertions correlate with several oxidative stress parameters have also been analyzed. Methods: We performed a longitudinal study based on the examination of T-lymphocytes isolated from the peritoneum of female mice without sacrificing them, allowing us to monitor each animal from adult to old age. Mitochondrial insertions into nuclear DNA were quantified by fluorescence in situ hybridization (FISH) and their chromosomal location was analyzed. Their possible implication in chromosome segregation failure and genetic loss were explored using the CBMN (Cytokinesis-Block Micro Nucleus assay) assay, which allows in vitro analysis of chromosome loss in binucleated lymphocytes with inhibited cytokinesis, in combination with FISH, using a mouse pancentromeric probe. The proliferative capacity of lymphocytes was estimated by measuring 3H-thymidine uptake after Concanavalin-A stimulation in cultures of these cells. The redox parameters (glutathione reductase activity, oxidized glutathione, and malondialdehyde concentrations) were quantified spectrophotometrically and fluorometrically in the peritoneal cell suspension. Results: Our results show for the first time the presence of mitochondrial sequences in the nuclear genome of mouse T-lymphocytes that progressive increase with aging. Their chromosomal localization was found at the centromeres of all autosomes and the X chromosome. In the same individuals, binucleated T-cells with micronuclei showed a significantly increased frequency associated with age. Most of them were positive for centromere sequences, reflecting the loss of chromatids or whole chromosomes. The proliferative capacity of T- lymphocytes decreased with age as well as the glutathione reductase activity, while the oxidized glutathione and malondialdehyde concentrations exhibited a significant increase. Numtogenesis positively correlated with micronuclei frequency, whereas both parameters were negatively related to lymphocyte proliferation capacity. Besides, numtogenesis negatively correlated with antioxidant enzymatic activity (glutathione reductase) and positively with oxidant parameters (oxidized glutathione and malondialdehyde concentrations). Conclusions: Our study provides insights for a new approach to understanding immunosenescence. We propose a novel mechanism in which mitochondrial fragments, originated by the increased oxidative stress status during aging, accumulate inside the nuclear genome of T-lymphocytes in a time-dependent way. The main entrance of mitochondrial fragments at the pericentromeric regions may compromise chromosome segregation, causing genetic loss that leads to micronuclei formation, rendering aneuploid cells with reduced proliferation capacity. The novelty of this proposal is worth mentioning since, to date, studies regarding insertions of mtDNA in the nuclear genome related to aging are scarce and circumscribed to the descriptive level. Further research deciphering this new mechanism will be the subject of our future investigations.

P24 — Circulating Cell-Free DNA in Plasma as a Potential Biomarker of Biological Age. Relationship with the Redox State and Function of Immune Cells. Influence of Gender

Estefanía Díaz-Del Cerro¹, Mónica González-Sánchez¹, Irene Martinez De Toda¹, Manuel Lambea¹, Jaime Bartolomé¹, Mónica De La Fuente¹ (1. Complutense University Of Madrid — Madrid (Spain))

Background: The aging process is a natural and progressive process characterized by the generalized deterioration of the functions of the organism, which leads to the loss of homeostasis and, therefore, of health. Aging is associated with an increased risk of morbidity and mortality. Nevertheless, chronological age fails to be an accurate indicator of one individual’s health or remaining lifespan. Thus, there is a need to identify biomarkers of aging that can be used to determine the biological age or the rate of aging of an individual. The basis of the global deterioration associated with aging resides in the establishment of chronic oxidative and inflammatory stress (due to an imbalance between oxidative and inflammatory compounds and their defenses, in favor of the former) as age advances. Such oxidative stress causes damage to various cellular components, such as proteins, lipids, and nucleic acids, interfering with the normal function of cells and increasing cellular apoptosis. Because of cellular apoptosis, fragments of DNA (both nuclear and mitochondrial) are released outside from the cell and they can reach blood circulation. Supporting this idea, elderly individuals show higher levels of circulating cell-free DNA (ccfDNA) in plasma, a fact that has been associated with chronic systemic inflammation. Nevertheless, it is not known how the levels of ccfDNA relate to oxidative stress parameters in plasma as well as to the function of immune cells, which are essential in the clearance of apoptotic cells. Moreover, the influence of gender on these levels is also unknown. Objectives: The objective of the present study was to study the age and gender-related differences in ccfDNA levels in plasma obtained from 70 healthy men and women (23–80 years old). For this purpose, three genes were selected: one with a nuclear origin (β-globin) and two with a mitochondrial one (NADH and Cox III genes). Amplification and quantification of a region of each of these genes were performed by q-PCR. In addition, it was investigated the relationship among the levels of these ccfDNAs and several parameters of oxidative stress and immune function, and the biological age of each individual, calculated with the Immunity Clock. Results: We found an age-related increase in the levels of the three investigated DNA fragments in plasma both in men and women, increasing from the 50s onwards. Men showed higher levels of the β-globin and Cox III genes than women in every decade of age, while higher levels of the NADH gene were observed in men after the 50s decade. Interestingly, we show that the levels of the nuclear fragments (β-globin) negatively correlate with the antioxidant parameters glutathione reductase activity (p=0.05) and the concentration of reduced glutathione (GSH) (p=0.04). The levels of the mitochondrial fragments (NADH gene) also correlate negatively with the concentration of reduced glutathione (p=0.04), whereas the β-globin/NADH ratio correlates negatively with the concentration of oxidized glutathione (GSSG) (p=0.05) and GSSG/GSH ratio (p=0.02). Concerning the function of immune cells, we found out that the levels of the nuclear β-globin (p=0.004) and mitochondrial Cox III (p=0.05) fragments in plasma correlate negatively with the cytotoxic Natural Killer activity of immune cells. Moreover, the levels of β-globin (p=0.04) and the β-globin/NADH ratio (p=0.023) were found to relate with the biological age of each individual calculated with the Immunity Clock. Conclusions: The ccfDNA fragments from the β-globin, NADH, and Cox III genes investigated in the present study increase with aging, both in men and women, while men show higher levels of these fragments than women. In addition, the levels of these fragments in plasma negatively correlate with the antioxidant defenses and with the cytotoxic activity of immune cells. This seems to support the idea of these fragments arising and accumulating due to an oxidative stress situation. Interestingly, the levels of the nuclear β-globin fragments in plasma have the potential to be used as a marker of biological age.

P25 — Identification of a Blood Micrornas-Based Signature of Ageing in Down Syndrome

Cristina Morsiani¹, Salvatore Collura¹, Maria Giulia Bacalini², Claudio Franceschi³, Miriam Capri¹ (1. Department Of Experimental, Diagnostic And Specialty Medicine, University Of Bologna — Bologna (Italy), 2. Irccs Istituto Delle Scienze Neurologiche Di Bologna — Bologna (Italy), 3. Laboratory Of Systems Medicine Of Healthy Aging And Department Of Applied Mathematics, Lobachevsky University — Nizhny Novgorod (Russian Federation))

Background: People affected by Down Syndrome (DS) undergo a premature ageing process with early onset of age-related disease. DS represents a model for the study of ageing acceleration and its biomarkers. In fact, different biomarkers of biological age have been analyzed in DS individuals, showing that DS individuals are biologically older than expected. MicroRNAs (miRs), a class of small non-coding RNA with a regulation role at post-transcriptional level, have been proposed as further epigenetic biomarkers in DS. Objectives: The main endpoint of this study was the identification of peripheral blood circulating miRs signatures characterizing DS phenotype and ageing process. Methods: A discovery phase based on arrays for the simultaneous analysis of 754 miRs in plasma samples, obtained from 3 young DS donors (31±2 years-old) and 3 elderly DS donors (66±2 years-old) was performed. A second phase of validation was carried out for all miRs with relevant fold changes between the two groups by RT-qPCR, then the validation was extended to an enlarged cohort of 44 DS individuals with different ages (from 19 up to 68 years-old) and sex (24 males and 20 females). A group of 30 healthy sex-age matched subjects was adopted as reference for the level of circulating miRs in peripheral blood, representative of the physiological ageing. A bioinformatics analysis was also performed on the identified miR-targets. Results: Four miRs have been identified i.e., miR-152-3p, miR-28-5p, miR-628-5p and let-7d-5p. Young DS (<50 years) showed more miRs changes than healthy age-matched control group, while no differences were revealed comparing elderly cohorts. To further investigate the role of the identified miR signature in ageing and DS, a bioinformatic analysis was performed on the validated targets of the identified miRs. Insulin-like Growth Factor 1 Receptor (IGF1R), known to be a key molecule for longevity, was found to be a validated target of all four miRs. Another interesting common target, Sigma-1 Receptor (SIGMAR1) was identified. This receptor has a role in maintaining cellular homeostasis and neuronal plasticity in the brain under physiological conditions and has a role in protecting the brain against cell loss due to injury or disease. Conclusion: MiR-628-5p, miR-152-3p, miR-28-5p and let-7d-5p may represent a circulating miRs signature for the characterization of the accelerated ageing in DS individuals. All the identified miRs appear to be down-regulated in young DS subjects compared to age-sex-matched healthy controls, but miR-28-5p and let-7d-5p assume a specific sign of a premature ageing compared with physiological ageing. To understand the underpinning mechanisms of miR-dysregulation may pave the way for translational therapeutic approaches aimed at slowing-down the ageing process in DS persons. Conflict of interest. The authors declare no conflict of interest.

P26 — Allostatic Load and Physical Performance: Findings from Imias Study

Ricardo Guerra¹, Matheus Germano¹, Crisitiano Gomes¹, Juliana Fernandes² (1. Federal University Of Rio Grande Do Norte — Natal (Brazil), 2. Federal University Of Pernambuco — Recife (Brazil))

Introduction: Allostatic load is related to cumulative adaptations to stressful situations, and can have deleterious effects on the health of individuals. The aim of the study is to investigate the relationship between physical performance, assessed by the Short Physical Performance Battery (SPPB) and allostatic load in community dwelling older adults. Methods: Data were collected from the baseline survey of the International Study of Mobility in Aging (IMIAS) realized in 2012, in different cities Kingston (Canada), Saint-Hyacinthe (Canada), Manizales (Colombia) and Natal (Brazil), the data were obtained from 1.102 older adults of both sex male (n=515) and female (n=587), with aged between 65 and 74, community residents. Allostatic load was assessed using 9 biomarkers divided into 4 subgroups, metabolic (glycated hemoglobin, HDL, triglycerides and albumin), immunological (interleukin 6 and C-reactive protein), cardiovascular (systolic and diastolic blood pressure) and anthropometric (mass index body), these biomarkers made the AL index ranging from 0 to 9. Physical performance was evaluated using the SPPB. In addition, sociodemographic, clinical, lifestyle and physical performance variables were evaluated. Results: Descriptive data show that there was no difference between cities in variables of age and allostatic load. However, SPPB scores and chronic conditions have differences between cities. The final linear regression model consisted of variables such as sociodemographic, clinical, lifestyle, allostatic load and physical performance measures, with the last variable dependent. The results revealed that low values of short physical performance battery are associated with independently higher allostatic load values (β: -0.247, Standard error: 0.034, p-value <0.001). Furthermore, significant associations were observed with chronic conditions (β: 1.443, Standard error: 0.207, p-value <0.001) and age showed an inverse relationship (β: -0.056, Standard error: 0.020, p-value = 0.004). Conclusion: In summary, we conclude that allostatic load is inversely associated with poor physical performance in older adults.

P27 — Unravel Immunosenescence Role in Response to Kidney Injury: A First Step Immune Landscaping

Snigdha Nitin Rao¹, Audrey Casemayou¹, Marie Buléon¹, Guylène Feuillet¹, Elodie Riant ¹, Alexia Zakaroff-Girard¹, Frédéric Martins¹, Ignacio Gonzalez-Fuentes¹, Jean Sébastien Saulnier Blache¹, Joost Peter Schanstra¹, Julie Belliere¹ (1. Institute Of Cardiovascular And Metabolic Diseases — Toulouse (France))

Background: Acute kidney injury (AKI) has been reported to account for 13.3 million cases worldwide and 1.7 million deaths per year. Clinical studies have shown that the elderly patients are at higher risk of AKI and have poorer outcomes after an episode of AKI. An incidence of 22% in the >80 age group and a mortality rate of 20% was reported. Among causes of AKI, rhabdomyolysis (RM) (which accounts for 10% of AKI cases) is usually associated with poor outcome especially when associated with multiple organ failure. Due to damage of the skeletal muscle, RM results in the release of myoglobin and toxins into the bloodstream, leading to AKI, renal failure, and potentially death. RM induced AKI (RIAKI) occurs frequently in elderly due to increased fall risk, RM-inducing drug exposures (statins) and worsening factors (dehydration, diarrhea). Immune system has been reported to be implicated in the pathogenesis of RIAKI, as well as inflammatory and previously described oxidative pathways. Macrophages have been especially reported to play a major role in the pathogenesis of RIAKI. Two macrophages subtypes (inflammatory R1 type and resident R2 type) have been described, and R1 have been reported to drive RIAKI lesions and prognosis. Pharmacological depletion of macrophages has been associated with an improved early and late renal and global prognosis in a murine model. Neither the role of other immune cells than macrophages nor the susceptibility of the immune response to be modified by aging were explored. Objectives: The main objective of this study was to describe immune cells recruitment during RIAKI and associated pathways through single cell RNA sequencing in young mice. The second objective of this study was to analyze the feasibility of RIAKI model in old mice to further perform immune cells comparison and explore immunosenescence pathways. Methods: In this study, 2 months (young) C57BL/6J male mice were used. Mice were either administered saline (control) or glycerol i.m. (7.5 ml/kg 50% glycerol diluted in saline) to induce RM. Blood was collected at the mouse tail vein blood at 6 hours, 24 hours and 48 hours and serum was obtained after centrifugation (5 minutes at 2000 rpm). RM intensity was measured through creatinine phosphokinase (CPK) dosage and renal function was assessed through blood urea nitrogen (BUN) concentrations measured on Pentra 400 analyzer. To describe immune cells recruitment in kidneys, the kidneys from control and glycerol-treated mice were decapsulated, minced and incubated with collagenase and DNase. Dissociated cells were then incubated with red cell lysis buffer and finally passed through a 40 µm filter. Dissociated cells were then incubated with CD45 antibodies and single cell sorting was performed was performed using BD Influx cell sorter. Single cell libraries were prepared and single cell RNA sequencing was performed according to 10X Genomics protocol. In the second study, we compared 2 (young) and 18 months (old) C57BL/6J male mice. Results: — Immune landscaping of kidney after RIAKI. Single cell RNA sequencing of CD45+ renal cells revealed 8 clusters which were further identified as macrophages, monocytes, T cells, B cells, neutrophils, NK cells, dendritic cells and endothelial cells. The repartition of cells was drastically modified by RMAKI with enrichment in macrophages, monocytes and neutrophils. Differential expression analysis between control and RIAKI group revealed upregulation of AA467197 (a long non-coding RNA) in multiple clusters, which is known to be upregulated in renal ischemia-reperfusion. NF-kB signaling pathway was upregulated as well in multiple clusters. Klf2 a transcription factor acting as an inducer of inflammation associated with NF-kB pathway was observed as a cluster specific gene observed in the B cell cluster. Subclustering of macrophages revealed for the first time macrophages diversity when identifying 7 clusters, sharing consensual markers for macrophages subtypes (M1, M2) and pro-inflammatory markers. Drug repositioning studies through connectivity map studies showcased SRC inhibition as a promising strategy. — RIAKI in old mice. Old mice responded to RIAKI model, in a similar extent that young mice. CD45+ cell sorting was performed in the old control group and is in process for the old RM group. Conclusion: This study showcased the immune landscaping in RIAKI in young mice and that the model is suitable for a further comparison with old mice. Future experiments are needed to compare the immune landscape of young mice with that of old mice through single cell RNA sequencing and this would further be used to identify immunosenescence targets and new therapeutic targets for rhabdomyolysis.

P28 — Leukocyte-Derived Ratios are Associated with Early-Onset Dementia and Functional Frailty

Yu Na Kim¹, Saleena Arif¹ (1. Dothousehealth — Boston (United States))

Background: Dementia consists of late-onset (LOD) and early-onset dementia(EOD)(age <65). Studies show leukocyte-derived-ratios could represent inflammation markers for cognitive decline in LOD. However, the relevance of these markers in EOD and functional frailty is not well studied. This retrospective study aims to evaluate the relation of inflammation markers with EOD and functional frailty in community-dwelling patients aged ≥55 seen at Cognitive Assessment Clinic 01/2021–08/2021 at our Community Health Center. The study included 63 patients with mean age(±SD) of 71.3±8.97, with 13 pre-geriatric (PG) patients aged < 65, further categorized by by sex: women=52, men=11, preferred language: Vietnamese=38, English=15, Spanish=7, others=3 and ethnicity: Asian=39, White=8, Black=13, unknown=3. In this study, we do not have a control group. Using standardized cognitive assessment tests, we diagnosed 37, 19, 7, and 5 patients with dementia, MCI(Mild Cognitive Impairment), EOD, and early-onset MCI(EOMCI), respectively. Seven patients(PG=1) had no cognitive impairment. We obtained ADLs(activities of daily living) and IADLs(instrumental activities of daily living) for functional assessment. 9(PG=5), 36(PG=8), and 18(PG=0) patients were functionally independent, independent in ADLs only, and dependent in ADLs and IADLs, respectively. We collected complete blood count (CBC) and calculated lymphocyte-to-monocyte ratio(LMR), neutrophil-to-lymphocyte ratio(NLR), and platelet-to-lymphocyte ratio(PLR) for 59 patients. Four patients had no data. The markers for dementia, no dementia, MCI and normal results cases are neutrophils (4589.09±1745.70, 4378.92±1574.13, 4295.33±1517.78, 4621.67±1862.80), lymphocytes(2389.71±799.93, 2165.5±789.32, 2204.56±635.69, 2048.33±1212.44), NLR(2.23±1.57, 2.18±0.95, 2.02±0.66, 2.68±1.51), monocytes(649.97±206.45, 591.58±210.78, 569.89±193.36, 656.67 ± 265.546), LMR (3.98±1.81, 3.93±1.58, 4.21±1.66, 3.09±1.01), eosinophils(256.02±183.51, 167.91±117.47, 173.89±127.00, 150±90.11), hemoglobin(13.18±1.07, 12.85±1.22.9, 12.76±1.23, 13.13±1.24), platelets(256.19k±65.51, 259.62k±72.18, 270.17k±72.27, 228k±67.89) and PLR(0.12±0.05, 0.13±0.05, 0.13±0.06. 0.13±0.03). For ① EOD vs no dementia in younger adults, and ② EOD vs EOMCI, the parameters are: neutrophils(① 4500.6±2312.84,4566.67±1273.84, p = 0.956, ② 4500.6 ± 23 12.84, 4460±1394.0, p = 0.974), lymphocytes(① 2699.6±1 159.87, 1816.67 ± 556.48, p = 0.174, ② 2699.6±1159.87, 1960 ± 482.7, p = 0.242,), NLR(① 1.92±1.43, 2.67±1.04, p=0.36, ② 1.92±1.43, 2.28±0.45, p=0.615), monocytes(① 477.6±181.96, 483.33±172.24, p = 0.959 ② 477.6±181.96, 480±192.4, p = 0.984), LMR(① 6.22±2.76, 4.40±2.67, p=0.299 ② 6.22±2.76, 4.84±2.73, p=0.45), eosinophils(① 217.8±129.65, 196.67 ± 202.65, p = 0.839 ② 217.8± 129.65, 228±209.7, p = 0.929), Hemoglobin(① 13.66±0.42, 13.62±1.34, p = 0.947 ② 13.66 ± 0.42, 13.32±1.3, p=0.603), platelets(① 301.14k±91.10, 288k±90.44, p=0.817 ② 301.14k±91.10, 315.2k±68.4, p=0.79) and PLR(① 0.11±0.05, 0.16±0.05, p=0.134 ② 0.11 ± 0.05, 0.17±0.06, p=0.125). The markers for ① functional independence vs dependence and ② dependence in only IADLs vs ADLs and IADLs are: neutrophils(① 4395.1 ± 1261.1, 4522.16±1740.3,p = 0.798, ② 4618.2±1893.2, 4351.4±1464.6, p=0.582), lymphocytes(① 2241.3±856.0, 2308.8±794.2, p=0.83, ② 2310.9±808.2, 2305±791.7, p = 0.98), NLR(① 2.24±1.12, 2.21 ± 1.39, p = 0.944, ② 2.26±1.6, 2.13±1.04, p=0.73), monocytes(① 552±168.3, 639.58±213.5,p=0.192, ② 605.7±201.1, 699.8±227.3. p=0.153), LMR(① 4.4±2.2, 3.9±1.6, p=0.49, ② 4.1±1.8, 3.42±1.08, p=0.102), eosinophils(① 175.6±174.0, 228.2±163.5, p=0.418, ② 202.3±147.2, 274.3±184.4, p=0.166), Hemoglobin(① 12.9±1.7, 13.1 ± 1.0, p=0.74, ② 13.2±1.0, 12.9±1.0, p=0.315), platelets(① 259.7k±96.7, 257.2k±62.6, p=0.942, ② 264.82k±66.92, 242.72k±52.12, p=0.202) and PLR(① 0.13±0.06, 0.12±0.05, p=0.647, ② 0.12±0.05, 0.11±0.04, p=0.443). High lymphocyte counts and low PLR are associated with EOD diagnosis, and high monocyte counts are related to a diagnosis of functional frailty. LMR can be considered a marker to detect EOD and functional frailty, and eosinophil and platelet counts are potential markers to discover functional dependence. The causality and predictive values of the inflammatory markers to diagnose EOD and functional frailty need to be investigated.

P29 — Does Inflammation Contribute to Cancer Incidence and Mortality During Aging? A Conceptual Review

Florent Guerville¹, Maël Lemoine¹, Victor Appay¹ (1. Immunoconcept Lab, Bordeaux University — Bordeaux (France))

Aging is associated with chronic low-grade inflammation on the one hand, and with cancer incidence and mortality on the other hand. As inflammation contributes to cancer initiation and progression, one could hypothesize that age-associated chronic low-grade inflammation contributes to the increase of cancer incidence and/or mortality observed during aging. Here we review the evidence supporting this hypothesis: (1) epidemiological associations between biomarkers of systemic inflammation and cancer incidence and mortality in older people, (2) therapeutic clues suggesting that targeting inflammation could reduce cancer incidence and mortality, and (3) experimental evidence from animal models highlighting inflammation as a link between various mechanisms of aging and cancer initiation and progression. Despite a large body of literature linking aging, inflammation and cancer, convincing evidence for a clear implication of specific inflammatory pathways explaining cancer incidence or mortality during aging is still lacking. Thus, we discuss how research could fill these gaps in evidence, and how such advances could be translated in clinical care.

P30 — Associations of Adverse Childhood Experiences with Executive Function and Brain-Derived Neurotrophic Factor in Older Adults

Cindy Tsotsoros¹, Misty Hawkins¹ (1. Oklahoma State University — Stillwater (United States))

Adverse childhood experiences (ACEs) may predict markers of neurocognitive performance (i.e., executive function; EF) and brain health/plasticity (i.e., brain-derived neurotropic factor; BDNF). This pilot examined the magnitude of effects between: 1) ACES and EF performance, 2) ACEs and BDNF levels, and 3) EF performance and BDNF levels in older adults. We hypothesize that higher ACEs will be associated with poorer EF scores and lower BNDF levels and that lower EF scores will be associated with lower BDNF levels. Given the pilot nature of the study, an emphasis is placed on effect size vs. significance. Participants were 55 older-aged women (age=73 years, BMI=30, racially minoritized=11%). ACES were quantified using the 10-item Adverse Childhood Experiences Scale. EF was measured using the Stroop Color Word test. BDNF was estimated using proBDNF and matureBDNF levels estimated from serum collected via venipuncture. Higher ACEs levels were associated with EF scores (b = 0.22, p = .18) but not statistically significant; higher ACEs showed a meaningful beta coefficient with proBDNF levels (b = 0.38, p = .009); but did not show an association with matureBDNF levels (b = 0.09, p = .50). EF scores and matureBDNF showed a negative coefficient that did not reach significance (b = -.25, p = .132), but EF scores and proBDNF did not have a meaningful association (b = -0.10, p = .57). In a modest pilot of older-age women, higher ACEs were associated with higher proBDNF, indicating greater adversity in childhood is linked to higher levels of precursor protein for neurotrophins levels in late adulthood. This study is ongoing, and a larger sample will have more power given the size of the detected coefficients.

LP11 — The Mediating Role of GDF-15 in the Association Between PA and Bodyweight in Older Adults

Jérémy Raffin¹, Geetika Aggarwal², Andrew D Nguyen², John E Morley², Sophie Guyonnet¹, Yves Rolland¹, Bruno Vellas¹, Philipe De Souto Barreto¹ (1. Gérontopôle De Toulouse, Institut Du Vieillissement, Centre Hospitalo-Universitaire De Toulouse, — Toulouse (France), 2. Division Of Geriatric Medicine, Saint Louis University School Of Medicine, — Saint Louis (United States))

Background: Late-life aging determines a typical bodyweight reduction that has been partly attributed to the so-called “anorexia of aging”. Physical activity (PA) plays a major role in preventing age-related body mass changes but the molecular mechanisms underlying its benefits remain elusive. GDF-15, a stress signaling protein involved in a myriad of age-associated physiological processes, has been the focus of intense research for its aversive role on food consumption and its negative relationship with long-term PA. Objective: The present study investigated the mediating effect of GDF-15 on the association between PA and body weight changes in older adults. Methods: One thousand eighty-three healthy adults aged 70 years and over who participated in the Multidomain Alzheimer Preventive Trial (MAPT) were included. PA volumes were assessed using questionnaires at baseline, 6 months, 1-year, 2-year, and 3-year; while plasma GDF-15 was measured at 1-year throughout the MAPT intervention. Multiple linear regressions were performed to test the association between the first-year mean PA volume and the 1-year GDF-15 levels. Linear mixed model regressions were conducted to explore the associations of the first-year mean PA volume and GDF-15 levels obtained at 1-year with subsequent bodyweight changes. Mediation analyses were performed to investigate whether GDF-15 mediate the association between mean PA volume and consecutive bodyweight changes. Results: Our analyses demonstrated that higher volumes of first-year mean PA predicted lower GDF-15 at 1-year, which in turn predicted lower bodyweight loss 2 years later. Conclusion: This study suggests that long-term PA may prevent the “anorexia of aging” by attenuating the age-related GDF-15 increase. Long-term interventional investigations are required to confirm the present associations. The authors declared no conflict of interest.

LP12 — Analysis of Secretome Composition of Senescent Human Vascular Smooth Muscle Cells and its Influence on T Cells Functions

Agata Gluchowska¹, Dominik Cysewski², Paulina Podszywalow-Bartnicka³, Malgorzata Sliwinska⁴, Piotr Sunderland⁵, Ewa Kozlowska⁶, Michal Dabrowski⁷, Ewa Sikora⁵, Grazyna Mosieniak⁵ (1. Laboratory Of Molecular Basis Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland), 2. Mass Spectrometry Laboratory, Institute Of Biochemistry And Biophysics, Polish Academy Of Sciences — Warszawa (Poland), 3. Laboratory Of Flow Cytometry, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warszawa (Poland), 4. Laboratory Of Imaging Tissue Structure And Function, Nencki Institute Of Experimental Biology Polish Academy Of Sciences — Warszawa (Poland), 5. Laboratory Of Molecular Basis Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warszawa (Poland), 6.Department Of Immunology, Institute Of Zoology, Faculty Of Biology University Of Warsaw — Warsaw (Poland), 7.Laboratory Of Bioinformatics, Neurobiology Centre, Nencki Institute Of Experimental Biology Of Polish Academy Of Sciences — Warszawa (Poland))

Background: Aging is an inevitable process that results in progressive loss of physiological integrity and increased morbidity. One of the processes that facilitate aging of the organism and promote age-related diseases is cellular senescence. It is caused by gradual or rapid accumulation of unrepairable DNA damage that lead to permanent growth arrest of the cell. Atherosclerosis, a common age-related disease, is a pathologic process occurring within the artery and is characterized by a complex interaction between vessel cells and immune cells leading to local inflammation and atherosclerotic plaque formation. Vascular smooth muscle cells (VSMCs) are the most abundant cell type in the arterial wall and it was demonstrated that during progression of atherosclerosis, VSMCs undergo senescence and secrete increased amount of soluble factors. This feature of senescent cells known as senescence associated secretory phenotype (SASP) significantly influences tissue microenvironment and favor proinflammatory state. Apart from soluble factors, senescent cells secrete also an increased number of extracellular vesicles (EVs), which play an important role in intercellular communication and participate in many physiological but also pathological processes. Objectives: Thus, the aims of our studies were (i) to performed detailed unbiased proteomic analysis of both soluble factors and EVs secreted by senescent VSMCs and (ii) to investigate the influence of SASP factors on T cells activation and differentiation. Methods: To investigate the role of EVs secreted by senescent VSMCs we took advantage of two experimental setups. In the model of stress induced premature senescence (SIPS) human VSMCs were treated with a single dose of H2O2 and cultured for subsequent 7 days. In the alternative model, the cells were cultured until they lost the replication potential and underwent replicative senescence (RS). Extracellular vesicles secreted by VSMCs were isolated from serum-free cell culture medium by ultracentrifugation after 24 hours of conditioning and then unbiased proteomic analysis using tandem mass tags (TMT)-based mass spectrometry of secreted proteins (both soluble and vesicular) was performed. Thereafter, we analyzed the influence of EVs on T cells activation and differentiation. Human T cells were isolated from buffy coats of healthy donors, activated using the anti-biotin particles loaded with CD2, CD3 and CD28 antibodies and cultured in the presence of i) conditioned medium (medium obtained after 24h culture of control or senescent VSMCs), ii) medium containing only soluble factors, devoid of EVs (EV-free medium) or iii) fresh medium supplemented with EVs secreted by control or senescent cells. The percentage of cells expressing a given activation marker was measured after 24 hours of culture using flow cytometry. To analyze the effect of EVs on secretion of cytokines by T cells, after 72 hours medium from the lymphocytes culture was collected and the amount of secreted cytokines (IL-17A, IL-10, INFγ, TNFα, IL-4, IL-2) was measured using Cytometric Bead Array (CBA). Results: We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs) comparing to the number of EVs secreted by control (proliferating) cells. Based on the unbiased proteomic analysis of VMSC-derived EVs more than 900 proteins were identified. Among significantly increased proteins, 30% were common to EVs secreted by both types of senescent cells, while almost two times more proteins were upregulated only in EVs secreted by SIPS cells. More than 40% of proteins, the level of which decreased in senEVs, were common to EVs secreted by cells undergoing either type of senescence (SIPS and RS). Among all identified proteins we were able to distinguish those, which were significantly increased in EVs secreted by senescent VSMCs and belonged to GO Biological Processes related to immune response and immune activity. Further research on T cells revealed that senescent cell conditioned medium depleted with EVs lowered the level of expression of activation markers on T cells. Moreover senEVs increased secretion of IL-17, INFγ and IL-10 by T cells. Conclusion: Our studies revealed that senescent VSMCs produce high number of EVs that have distinct proteomic composition. EVs secreted by senescent VSMCs affect T cell functioning by modulating their activity and cytokine production. Thus senEVs might promote inflammatory response of the immune cells, which could have a tremendous significance for atherosclerotic plaque development. This work is supported by grant National Science Centre UMO 2014/15/B/NZ3/01150.

LP13 — Comparison of Human Vascular Smooth Muscle Cells Undergoing in vitro or in vivo Senescence and Analysis of the Influence of Their Sasp on the T Cells Functions

Agata Głuchowska¹, Małgorzata A. Sliwińska², Dorota Janiszewska³, Krzysztof Bojakowski⁴, Ewa Kozłowska⁵, Ewa Sikora³, Grażyna Mosieniak³ (1. Laboratory Of Molecular Basis Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warszawa (Poland), 2. Laboratory Of Imaging Tissue Structure And Function, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland), 3. Laboratory Of Molecular Basis Of Aging, Nencki Institute Of Experimental Biology, Polish Academy Of Sciences — Warsaw (Poland), 4. 2nd Vascular Department, Center Of Postgraduate Medical Education — Warsaw (Poland), 5. Department Of Immunology, Institute Of Functional Biology And Ecology, Faculty Of Biology University Of Warsaw — Warsaw (Poland))

Background: Cellular senescence is a highly dynamic and complex process, during which the properties of senescent cells continuously evolve and change from early to late senescent state. An excellent example of an age-related disease, which development depends on senescence is atherosclerosis. Senescent vascular smooth muscle cells (VSMCs) have been identified within the atherosclerotic plaques (AP). Moreover, it was shown that senescent VSMCs contribute to the plaque development mainly due to diminished proliferation potential that limits efficient plaque repair as well as the secretion of SASP factors. Those factors may favor proinflammatory state of atherosclerotic plaque by actively modulating neighboring microenvironment and affecting immune cells. However detailed analysis of senescent cells present in the plaque has certain limitation. Objectives: The aims of our studies were (i) to visualize and compare morphology, ultrastructure and expression of senescence markers in early and late senescent VSMCs and senescent VSMCs derived from human atherosclerotic plaques; (ii) to analyze and compare the secretory profile of VSMCs that underwent process of senescence under in vitro or in vivo condition during plaque formation using cytokine arrays (iii) to investigate the influence of SASP factors on T cells activation, proliferation and migration. Methods: In our research we used two models of senescence of VSMCs: stress induced premature senescence (SIPS), during which cells were treated by hydrogen peroxidase and culture for 7 days — defined by us as an early state of senescence (SIPS I) or culture for 4 weeks, i.e. late state of senescence (SIPS IV). Alternatively, cells were cultured until they lose the replication potential and underwent replicative senescence (RS). Both, SIPS and RS represent a process of in vitro senescence. For studies of in vivo model of senescence we used VSMCs isolated form atherosclerotic plaques (AP), derived from the carotid artery of patients. To visualized and characterize ultrastructure of senescent cells we used transmission and scanning electron microscopy. Secretory profile of senescent cells was analyzed using cytokine antibody arrays. We measure the level about 130 cytokines e.g. growth factors, metalloproteinases, chemokines and inflammatory factors present in the conditioned medium and performed comparative analysis of those factors secreted by VSMCs undergoing in vitro and in vivo senescence. Thereafter, we analyzed the influence of SASP factors on T cells activation, proliferation and migration. Human T cells were isolated from buffy coats of healthy donors, activated using the anti-biotin particles loaded with CD2, CD3 and CD28 antibodies and cultured in the presence of medium obtained after 24h culture of control or senescent VSMCs (both undergoing process of senescence under in vitro or in vivo condition). Using flow cytometry we measured the expression of markers of activation (CD25, CD69 and CD38) and characterized proliferation of CD4+ and CD8+ T cells. Results: Analysis of SA-β-gal revealed that senescent VSMCs (SIPS I, SIPS IV, RS and AP) differ in the level of SA-β-gal. We also observed that AP-derived cells showed higher level of BrdU and Ki67 positive cells then cells undergoing SIPS or RS. Scanning electron microscopy observation revealed analogous changes in the morphology of each type of senescent cells however the most remarkable changes were noticed for SIPS cells. More detailed analysis based on transmission electron microscopy let us to observe elongated and damaged mitochondria, expanded endoplasmic reticulum and accumulation of enlarged and dysfunctional lysosomes present in VSMCs undergoing in vitro and in vivo senescence. Analysis of secretory profile revealed that the level of 20% of cytokines did not change in both, control and senescent cells. The level of 39 cytokines increased in all types of senescence, and the level of only 8 cytokines decreased relative to control. There were also cytokines, the level of which has changed only in cells derived from the plaques. What was interesting our data indicates that cytokines secreted by plaque-derived VSMCs largely resembles the secretory profile of replicative and late state of premature senescent cells. Thereafter, we showed that SASP factors secreted by VSMCs undergoing in vitro senescence promoted activation of T cells. This effect was not observed for VSMCs derived from AP. On the other hand SASP components secreted by AP-derived, but also late state senescent VSMCs, stimulated T cells proliferation the most efficiently. What is more, SASP factors secreted by VSMCs undergoing both in vitro and in vivo senescence promoted migration of T cells, however factors secreted by AP-derived cells had the biggest impact. Conclusion: Our studies revealed the similarities and differences between VSMCs undergoing in vitro and in vivo senescence. Although we were able to identified common features for each type of senescence some differences concerning SASP profile do exist. Those differences might determine the role of senescent VSMCs present within the plaque. Thus we should be aware about certain discrepancy between the data obtained from studies of senescence performed under cell culture conditions. This work is supported by National Science Centre grants UMO 2014/15/B/NZ3/01150 and OPUS UMO-2018/31/B/NZ3/02931

LP14 — Roles of Senescence and Inflammation in Noonan Syndrome Pathophysiology

Laurène Mazeyrie¹, Ophélie Pereira¹, Lucas Bourdens¹, Marie Sallese¹, Céline Saint-Laurent¹, Romain Paccoud¹, Marine Delagrange², Jean-Philippe Pradère¹, Philippe Valet¹, Cédric Dray¹, Thomas Edouard², Armelle Yart¹ (1. Restore, Umr1301 — Toulouse (France), 2. centre Hospitalier Universitaire De Toulouse — Toulouse (France))

Background: According to the WHO, the population aged 60 and over will have doubled by 2050. Unfortunately, aging comes along with an explosion of aging-associated disorders, such as metabolic and cardiovascular diseases, bone and muscle weakening, cognitive dysfunction, that all contribute to the loss of functional capacities, leading to frailty and dependence. The healthy aging is therefore fundamental from a societal and economic point of view, which requires to understand how aging drives these disorders. Intriguingly, several genetic disorders associate from infancy multiple features that are reminiscent of age-associated diseases, highlighting the single genetic event causing these conditions as a potent target during natural aging. In that context, Noonan Syndrome (NS), a relatively frequent (1/2000 live births) genetic disease associating multiple congenital defects (dysmorphic features, cardiopathies, short stature, developmental delay), seems to be also characterized by a premature aging phenotype. Indeed, patients with NS display musculo-skeletal defects (e.g. decreased bone mass and muscle weakness), predisposition to myeloproliferative disorders (MPD), and metabolic disturbances (e.g. insulin resistance). NS is mainly caused by gain-of-function mutations in PTPN11 gene, encoding the tyrosine phosphatase SHP2. They result in SHP2 hyperactivation, triggering an upregulation of the RAS/MAPK pathway, which has been causally linked to NS pathophysiology. Consistent with a premature aging phenotype, activation of oncogenes such as RAS (a SHP2 target) triggers senescence, a cellular stress and damage response that drives aging. Moreover, recent data reveal recurrent dysfunctions of myelomomocytic cells (macrophages, myeloid precursors, osteoclasts) in lane with NS clinical signs and the contribution of myeloid cells and an inflammatory component (the so-called inflammaging) in aging-associated diseases is well established. We thus hypothezise that NS could be a model of premature aging, at least in part through myeloid cell alterations, and that SHP2 hyperactivation may contribute to age-related disorders in general, highlighting a potential target to combat age-related diseases. Objectives: Our objectives are: I-To document the NS-associated inflammaging phenotype in an established mouse model of NS, with a particular focus on metabolic disorders and muscle weakness. II- To study the role of myeloid cells dysfunction on age-related disorders and their implication in the establishment of senescence. III- To decipher the role of cellular senescence on NS inflammaging like phenotype. Methods: I- We performed an extensive phenotyping of NS SHP2D61G/+ mice phenotype (metabolic imbalance by glucose/insulin resistance tests and analyses on metabolic tissues, muscle frailty by grip test and other functional tests followed by muscle analysis). II- The role of myeloid cells in these dysfunctions was assessed by bone marrow transplantation (BMT) in lethally irradiated mice. III- To determine whether and how increased senescence contributed to this phenotype, senescence was measured in different tissues/cells, and genetic reduction of the senescence process was achieved by the mean of p16INK4A gene invalidation in NS mice. Results: I- NS mice display insulin resistance and reduced muscle mass and strength. This phenotype correlates with an upregulation of inflammatory gene expression in liver, adipose tissue and muscle. Moreover, we find an overexpression of senescence markers, notably p16 in these same tissues. II- Repopulation of NS mice with WT bone marrow cells restores insulin sensitivity and increases muscle strength and normalizes p16 expression in the different tissues. III- Partial reduction of p16 expression rescues in part the phenotype of NS mice with a recovery of the glucose tolerance. However, muscle weakness isn’t improved and we didn’t observe alteration on inflammatory genes expression. Conclusions: These data reveal a premature aging phenotype, associating metabolic defects and muscle weakness, in a mouse model of NS. Both clinical traits are linked to myeloid cells dysfunction and increased senescence, highlighting a possible role of SHP2 hyperactivation in the onset of aging-associated diseases. In this context, ongoing experiments are directed to understand the interplay between myeloid cells dysfunction and senescence in NS. Beyond rare diseases, we will also determine whether and how SHP2 is dysregulated during aging and if it contributes to the development of age-associated diseases, such as insulin resistance and sarcopenia.

LP15 — Chromatin Structure Throughout Aging in Immune Cells (Immunosenescence) and in Cancer

Benoît Aliaga^1,2, Vera Pancaldi^1,2,3 (1. Centre De Recherches En Cancerologie De Toulouse — Toulouse (France), 2. INSERM — Toulouse (France), 3. Barcelona Supercomputing Center — Barcelona (Spain))

The western population is growing older and cancer risk increases as we age. This will become an even bigger major public health issue and studying the close relation between these two phenomena is a major challenge. In the first place, aging and cancer have opposite behavior. Aging is loss of cellular fitness. Cells become old, die and are not replaced by new cells because the old stem cells are less mobilized than young stem cells. On the other hand, cancer is a gain of cellular fitness. Cells become pre-cancerous and start to develop a benign tumor and, after some time, they become cancerous and multiply out of control. Surprisingly, these two phenomena share common origins. For example, they are both characterized by genomic instability, mitochondrial dysfunction, epigenomic alterations and so on. In cancer, one hypothesis postulates that the cancer immunosurveillance fails to recognize and kill the tumors. In the last few years, several papers pointed out the role of immunosenescence in cancer development. In other words, the aged immune system loses its plasticity and does not recognize tumor cells and cannot kill them. In literature, immunosenescence affects innate and adaptive immunity. For example, senescent T cells exhibit abnormal phenotypes (downregulation of CD27, CD28 and upregulation of CD57). More interestingly, some works showed that the tumor microenvironment (TME) can induce the senescence of immune cells and affect their functions. Despite the recent progress, the interaction between aging and cancer remains unclear. To explore this ambiguous relationship, the main question of my postdoctoral project is the following: how do the immunosenescent cells change their plasticity and identity in cancer patients? My main hypothesis is that the immune cells gain an altered chromatin structure when they become aged. At this poster, I will present the methods and datasets I will employ in this project and preliminary results of the computational analysis.

LP16 — Diet and the Hallmarks of Aging

Natasha De Franca¹, Yves Rolland^1,2, Sophie Guyonnet^1,2, Bruno Vellas^1,2, Philipe Barreto^1,2 (1. Gérontopôle Of Toulouse, Institute Of Aging, Toulouse University Hospital — Toulouse (France), 2. Maintain Aging Research team, CERPOP, Université de Toulouse, Inserm, Université Paul Sabatier — Toulouse (France))

Background: Geroscience is based on the hypothesis that basic mechanisms driving aging and age-related diseases overlap, what make them targets of interventions that could modify the rates of aging and delay the onset of diseases and disabled conditions. These interventions include dietary strategies such as dietary restriction, the intake of foods, extracts or supplements rich in bioactive compounds, and the adhesion to specific dietary patterns. Dietary restriction is usually characterized by the reduction of caloric intake (CR) and is the most well-known strategy experimentally leading to the extension in lifespan through evolutionary mechanisms and phenotypes. The potential role of bioactive compounds is based on their antioxidant activity, since an oxidative stress state is a common scenario in aging and can trigger degenerative and age-related chronic disorders. Finally, evaluating dietary patterns allows to assess the impact of the overall diet in the hallmarks of aging. Objectives: To discuss the literature evidence on the role of dietary restrictions, bioactive compounds, and dietary patterns as potential dietary strategies on the modulation of the nine hallmarks of aging. Methods: A narrative review was conducted. To avoid losing important studies in this field, a search in the PubMed database was performed in November 2021, by combining keywords of each hallmark of aging with keywords of each one of the three dietary domains, totaling 27 searches. To strict the search in some domains, we additionally included (AND) “aging” OR “lifespan” in the strategy. No restriction of publication data or language was made. The search strategy retrieved 877 manuscripts, with most of them focusing in the relationship between diet and deregulated nutrient sensing (N=319), followed by: 105 on genomic instability, 99 on cellular senescence, 94 on loss of proteostasis, 93 on telomere attrition, 60 on altered intercellular communication, 52 on mitochondrial dysfunction, 51 on epigenetic alterations, and only 4 on diet and stem cell exhaustion. Results: Dietary restriction, usually operationalized as 30% to 40% reduction in caloric intake, is the main strategy experimentally applied to study the axis diet-hallmark of aging. Promoting CR was demonstrated to modulate all the nine hallmarks, with most consistent evidence on genomic instability, loss of proteostasis, deregulating nutrient sensing, and cellular senescence, leading to increased life expectancy. However, most of the studies are restricted to cell cultures and animal models. Some concerns arise from the impact of CR on telomere attrition in humans, with this strategy leading to psychological stress and changes in diet composition associated with shorter telomeres and lower telomerase activity. The effect of bioactive compounds is even more limited in humans, especially considering the synergisms and the modality of administration and potential negative effects. In general, tea polyphenols, resveratrol, berries and wine extracts, and curcumin were found to be protective against deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, and altered intercellular communication. Both CR and bioactive compounds were also associated with improvements in clinical outcomes, especially glycemic homeostasis, blood pressure, and cognitive and memory outcomes. Much less evidence exists on the link between dietary patterns and the hallmarks of aging, with limited evidence suggesting that the adherence to dietary patterns source of phytochemicals and unsaturated fatty acids, such as the Mediterranean Dietary Pattern and other similar plant-based diets, might bring forth benefits on genomic instability, telomere attrition, and altered intercellular communication, albeit the results are limited to observational studies with specific populations. Conclusion: nutritional strategies are modifiable factors that can impact the hallmarks of aging, leading to potential changes in the rates of aging and the development of age-related clinical conditions. However, methodological limitations, such as the scarcity of investigations in humans, preclude currently the elaboration of more precise dietary recommendations aiming slowing down biological aging.

LP17 — Association Between Plasma Growth Differentiation Factor 15 and Blood Pressure Variability in Community-Dwelling Older Adults: The Mapt Study

Leonardo Bencivenga¹, Mathilde Strumia², Vellas Bruno³, Philipe De Souto Barreto³, Laure Rouch³ (1. Università Degli Studi Di Napoli Federico Ii — Napoli (Italy), 2. Centre Régional De Pharmacovigilance Et D’information Sur Les Médicaments — Toulouse (France), 3. Gérontopôle De Toulouse, Institut Du Vieillissement, CHU De Toulouse — Toulouse (France))

Background: Aging is associated with impairment in neuro-cardiovascular regulatory mechanisms, including those involved in Blood Pressure (BP) control. Increased Blood Pressure Variability (BPV) is related to organ dysfunction and may represent a sign of alteration in physiological homeostatic patterns, typical of late life. Furthermore, BPV exerts independent predictive value on typical geriatric syndromes, such as sarcopenia and frailty, and several health outcomes, especially cardiovascular diseases, neurocognitive decline, and metabolic disorders. Increased BPV also shares many molecular mechanisms with aging, such as low-grade inflammation common in advanced age, mainly through enhanced production of Reactive Oxygen Species (ROS) and endothelial dysfunction. In the field of Geroscience, growth/differentiation factor 15 (GDF-15) has emerged as one of the most promising biomarkers of inflammation, senescence and endothelial dysfunction. Accordingly, repeated evidence has reported associations between GDF-15, inflammatory conditions and mitochondrial stress in aging and age-related disorders (metabolic, cardiovascular, neurodegenerative). Objective: We aimed to determine whether baseline plasma GDF-15 levels are associated with visit-to-visit BPV in a population of community-dwelling older adults. We hypothesized that higher GDF-15 levels, indicative of increased inflammatory status and mitochondrial dysfunction, are associated with higher BPV, as plausible epiphenomenon of the aging process. Methods: This study constitutes a secondary analysis from the Multidomain Alzheimer Preventive Trial (MAPT). Participants were selected among non-frail community-dwelling volunteers aged ≥70 years who underwent repeated clinical examinations, including BP assessment. Plasma blood sample for GDF-15 was collected 12 months after enrolment and constituted our baseline. BP was assessed thereafter at 12, 18, 24, 30, 36, 48 and 60 months in a lying position at the brachial artery using a validated electronic device after at least 5 minutes of quiet rest. Systolic BPV (SBPV) and diastolic BPV (DBPV) were determined using several validated indicators: standard deviation (SD), coefficient of variation (CV), average real variability (ARV), successive variation (SV), variation independent of mean (VIM), and residual SD (RSD). Results: The study population consisted of 1055 elderly patients with mean age of 75,28 ± 4.36, predominantly female (693; 64,75%). Participants in the high GDF-15 group (according to the median) were older (76,45 ± 4,63 vs. 74,11 ± 3,7; p<0.0001), tended to be male (257: 47,33% vs. 137; 25,18%; p<0.0001) and to have higher Body Mass Index (BMI) (26,4 ± 4,18 vs. 25,66 ± 3.89; p<0.0027). They suffered significantly more from diabetes mellitus, ischemic heart disease, atrial fibrillation, chronic kidney disease and were more treated with antihypertensives agents. In the multivariate regression analysis, higher values of GDF-15 were significantly associated with increased SBPV (all indicators) after adjustment for demographics, BMI, MAPT randomization group, baseline systolic BP, use of antihypertensive drugs, diabetes mellitus, presence of cardiovascular and non-cardiovascular conditions [adjusted 1-SD increase for CV: β (SE)= 0.07 (0.03), p<0.04]. GDF-15 levels were not associated with DBPV [adjusted 1-SD increase for CV: β (SE)= 0.003 (0.03), p<0.93]. Conclusion: Higher GDF-15 levels were associated with higher SBPV. Our findings support the age-related processes of inflammation, senescence and endothelial dysfunction underlying BP instability, thus suggesting BPV to constitute a potential marker of aging. References: Rouch, L., et al. Visit-to-Visit Blood Pressure Variability Is Associated With Cognitive Decline and Incident Dementia. Hypertension 2020, 76, 1280–1288. Lattanzi, S., et al. Blood pressure variability and neurocognitive functioning. J. Clin. Hypertens. 2018. Chowdhury, E.K. et al. Systolic blood pressure variability is an important predictor of cardiovascular outcomes in elderly hypertensive patients. J. Hypertens. 2014, 32, 525–533. Rothwell, P.M., et al. Prognostic significance of visit-to-visit variability, maximum systolic blood pressure, and episodic hypertension. Lancet 2010 375, 895–905. Fujita, Y. et al. Secreted growth differentiation factor 15 as a potential biomarker for mitochondrial dysfunctions in aging and age-related disorders. Geriatr Gerontol Int 2016; 16, 17–29. Baek, S.J. et al. Growth differentiation factor 15 (GDF15): A survival protein with therapeutic potential in metabolic diseases. Pharmacol Ther. 2019, 198:46–58.

LP18 — Repurposing Antidiabetic Drugs for Rheumatoid Arthritis: Results from a Two-Sample Mendelian Randomization Study

Chenxi Qin¹, Lina Marcela Diaz-Gallo^2,3, Bowen Tang¹, Yunzhang Wang¹, Leonid Padyukov^2,3, Johan Askling^4,5, Sara Hägg¹ (1. Department Of Medical Epidemiology And Biostatistics, Karolinska Institutet — Stockholm (Sweden), 2. Division Of Rheumatology, Department Of Medicine Solna, Karolinska Institutet, Karolinksa University Hospital — Stockholm (Sweden), 3. Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. — Stockholm (Sweden), 4. Clinical Epidemiology Division, Department Of Medicine Solna, Karolinska Institutet, Stockholm, Sweden — Stockholm (Sweden), 5. Rheumatology, Theme Inflammation and Infection, Karolinska University Hospital, Stockholm, Sweden — Stockholm (Sweden))

Background: Rheumatoid arthritis (RA) patients have impaired glucose metabolism (1). Antidiabetic drugs like thiazolidinediones could reduce RA risk and even decrease disease activity and inflammation among non-diabetic RA patients (2–4). Objectives: We aimed to explore the repurposing potential of antidiabetic drugs for RA using Mendelian randomization (MR) to investigate the effects of genetically predicted glucose-lowering through different drug pathways on RA. Methods: MR uses alleles randomized at conception as genetic instruments (SNPs) to estimate unconfounded associations between an exposure and outcome and may thus be a viable method to gauge the potential of repurposing antidiabetic drugs for RA. This study selected six drug classes including glucagon-like peptide 1 receptor agonists (GPL-1 RAs), sodium/glucose cotransporter 2 (SGLT2) inhibitors, dipeptidyl peptidase IV (DPP4) inhibitors, insulin, thiazolidinediones and sulfonylureas. Genes encoding protein targets of antidiabetic drugs were identified from Drugbank and ChEMBL databases. Independent SNPs (linkage disequilibrium r2 < 0.01) from those gene regions were chosen as instruments. Since an effective response of antidiabetic drugs leads to lowered blood glucose, SNP-glucose associations for those instrumental SNPs were used to proxy the long-term pharmacological modulation of antidiabetic drugs. In addition, independent SNPs that reached the genome-wide significance (P <5e-8) instrumented the blood glucose level. Summary statistics were from genome-wide association studies on blood glucose (UK Biobank) and RA, respectively (5). The effects of genetically predicted glucose lowering on RA were estimated by the inverse-variance weighted or Wald ratio test. Sensitivity analyses included MR-Egger, weighted median and weighted mode methods. The intercept from the MR-Egger regression was tested to detect the horizontal pleiotropy. Results: A total of 69, 8, 2, 18, 18, 23 and 9 SNPs (linkage disequilibrium r2 < 0.01) were chosen as instruments of blood glucose, GPL-1 RAs, SGLT2 inhibitors, DPP4 inhibitors, insulin, thiazolidinediones and sulfonylureas, respectively. Overall genetically predicted glucose lowering was not associated with RA risk (odds ratio [OR]: 1.01 per 0.1 mmol/L decrease in glucose; 95% confidence interval [CI]: 0.97–1.05). However, lower genetically predicted glucose through thiazolidinediones (target gene: PPARG) was inversely associated with RA risk (OR=0.54; 95%CI, 0.35–0.83). Corresponding ORs and 95%CIs were 1.38 (0.97–1.95) for GLP-1 RAs, 0.13 (0.01–1.54) for SGLT2 inhibitors, 0.70 (0.45–1.10) for DDP4 inhibitors, 1.21 (0.82–1.78) for insulin and 1.21 (0.97–1.52) for sulfonylureas, respectively. Sensitivity analyses regarding thiazolidinediones were robust. The test of MR-Egger intercept concerning thiazolidinediones was not suggestive of horizontal pleiotropy (intercept=0.006 and P=0.884). Conclusion: Using MR analysis, we provide genetic evidence supporting thiazolidinediones, but not other antidiabetic drugs, as a potential drug class to reduce RA risk. References: 1. Ristic GG, Subota V, Stanisavljevic D, et al. Impact of disease activity on impaired glucose metabolism in patients with rheumatoid arthritis. Arthritis Res Ther 2021;23(1):95. DOI: 10.1186/s13075-021-02476-0. 2. Hsieh MS, Hung PS, Hsieh VC, Liao SH, How CK. Association between thiazolidinedione use and rheumatoid arthritis risk in patients with type II diabetes, a population-based, case-control study. Int J Clin Pract 2021;75(3):e13804. (In eng). DOI: 10.1111/ijcp.13804. 3. Marder W, Khalatbari S, Myles JD, et al. The peroxisome proliferator activated receptor-γ pioglitazone improves vascular function and decreases disease activity in patients with rheumatoid arthritis. J Am Heart Assoc 2013;2(6):e000441. (In eng). DOI: 10.1161/jaha.113.000441. 4. Ormseth MJ, Oeser AM, Cunningham A, et al. Peroxisome proliferator-activated receptor gamma agonist effect on rheumatoid arthritis: a randomized controlled trial. Arthritis Res Ther 2013;15(5):R110. DOI: 10.1186/ar4290. 5. Ishigaki K, Sakaue S, Terao C, et al. Trans-ancestry genome-wide association study identifies novel genetic mechanisms in rheumatoid arthritis. medRxiv 2021:2021.12.01.21267132. DOI: 10.1101/2021.12.01.21267132.

Microbiome and Aging

P31 — Late Onset Pharmacological or Dietary Interventions in Mice Improve Healthspan and Lifespan in Male and Female Mice

Mitchell Sarah¹, Michael Macarthur¹, Alice Kane², David Sinclai², Mehmet Huseyin³, Vath James³, Manning Brendan⁴, Mitchell James¹ (1. Eth Zurich — Zurich (Switzerland), 2. Harvard University — Boston (United States), 3. Zafgen — Boston (United States), 4. Harvard — Boston (United States))

Background/Objectives: While late-onset dietary or pharmacological interventions can extend longevity in rodents, whether or not they can be used to reverse or forestall onset of aging-related symptoms (i.e. frailty) remains untested. Here, we employed three interventions to test this hypothesis. Methods: Male and female C57BL/6 mice were randomized to one of four groups: control, 15% calorie restriction (15CR), 0.1% Methionine Restriction (MR) or ZGN1062 (1.5mg/kg, drug in feed) starting at 21mo. Healthspan measurements (mouse clinical frailty index (FI), blood collection, and hematology) were performed every three months. Fecal microbiome and immunophenotyping was performed. Survival was recorded for all mice. Results: At baseline there were no significant differences in frailty index. After 6mo FI increased consistent with reduced healthspan in control males (0.23±0.01 to 0.34±0.01 A.U, p<0.0001) and females at this age (0.19±0.03 to 0.24±0.01, p<0.0001). Male 15CR, MR and ZGN1062 mice had significantly lower FI scores at 27mo age (15CR: 0.32±0.01, p=0.02; MR: 0.31±0.01, p=0.0009; ZGN1062: 0.30±0.01, p<0.0001). Female mice were less frail than males at 27mo, suggesting sexual dimorphism in the timing of frailty onset in mice. ZGN1062 significantly extended lifespan in males (HR=0.56, p=0.007) and females (HR=0.46, p=0.001). There was a sexual dimorphism in the ability of 15CR and MR to extend lifespan, and a trend towards increased lifespan in males (HR=0.69, p=0.057 and HR=0.71, p=0.09) but not in females. Histological analysis for cause-of-death is ongoing. We identified novel sex and intervention specific associations between microbial communities and immune cells in old mice. Conclusion: Taken together these data suggest that a pharmacological intervention associated with weight loss, which may be a more practical therapeutic strategy towards mitigation of age-related healthspan decline than dietary restriction-based interventions.

LP19 — T Cell Metabolic Dysfunction Accelerates Intestinal Ageing

Manuel M. Gómez De Las Heras^1,2, Naohiro Inohara³, Mario Pérez-Manrique^1,2, Gabriel Núñez³, María Mittelbrunn^1,2 (1. Departamento De Biología Molecular, Centro De Biología Molecular Severo Ochoa, Centro Superior De Investigaciones Científicas-Universidad Autónoma De Madrid — Madrid (Spain), 2. Instituto de Investigación Sanitaria del Hospital 12 de Octubre (i+12) — Madrid (Spain), 3. Department Of Pathology, University Of Michigan — Ann Arbor, Michigan (United States))

Background: Demographic ageing is becoming a major burden due to the increase in multimorbidity and its age-related diseases. Therefore, the design of new strategies to foster healthier ageing is currently urging. The intestine is a physical and immunological barrier that harbours a microbial ecosystem termed gut microbiota, and whose age-associated deterioration could unleash the development of such pathologies. Specifically, host-microbiota symbiosis is lost during ageing due to a myriad of environmental and host intrinsic factors such as aberrant germinal centre (GC) responses, which support perturbations in the gut microbial communities driving gut dysbiosis. Along with a pro-inflammatory milieu, gut dysbiosis enhances intestinal permeability favouring the systemic dissemination of bacterial products. This event contributes to an age-related chronic inflammation (inflammageing), which is linked to cardiometabolic and neurologic disorders associated to ageing. Objectives: Previous data from the laboratory demonstrate that Cd4creTfamflox/flox mice, carrying a mitochondrial dysfunction in T cells, display inflammageing that results in multimorbidity (i.e., cardiovascular alterations and cognitive dysfunction), leading to a premature death. Thus, we hypothesised whether T cell metabolic failure leads to the loss of intestinal homeostasis accelerating ageing in this mouse model. Methods: Gut T cells were evaluated using flow cytometry and faecal bacterial communities were determined by 16S rRNA gene sequencing. Finally, intestinal barrier integrity was assessed by gut permeability tests, RT-qPCR and ELISA. Results: Our findings indicate that young Cd4creTfamflox/flox mice show altered intestinal T cell populations with increased markers of activation and senescence, as well as dysregulated GC responses compared to control mice. Accordingly, the analysis of Cd4creTfamflox/flox mouse faecal bacteria reveals a disrupted configuration of the gut microbiota. Finally, in vivo experiments support a premature alteration in the physical barrier function. Conclusion: Overall, our results suggest that mitochondrial dysfunction in T cells perturbs intestinal T cell subsets, affecting host-microbiome symbiosis and intestinal barrier integrity in this mouse model.

LP20 — Functional State of Patients with Osteoarthritis of HIP and KNEE Joints — A Pilot Study

Agnieszka Krawczyk-Wasielewska¹, Przemysïaw Keczmer¹, Ewa Mojs², Wïodzimierz Samborski¹ (1. Department Of Rheumatology, Rehabilitation And Internal Medicine, Poznan University Of Medical Sciences — Poznań (Poland), 2. Department Of Clinical Psychology, Poznan University Of Medical Sciences — Poznań (Poland))

Background: Osteoarthritis is the most common disease of the locomotor system. It is chronic and progressive, which significantly affects the functional state of patients, leading to reduced efficiency and hindering or preventing professional, family and social functions. Progressive degenerative changes in the joints lead to a reduction in the range of motion and weakening of muscle strength, which limits the activities of everyday life. For the purposes of planning the patient’s rehabilitation, we need not only the assessment of degenerated structures, but also the assessment of disturbances in the field of everyday activity. Because improving the functional state of patients is the main goal of rehabilitation in patients with osteoarthritis, it is difficult to imagine the evaluation of the progress of therapy without tools allowing to assess the functional state. Objectives: The aim of the study was to assess the functional status of patients diagnosed with osteoarthritis of the knee and hip joints based on the ICF classification and everyday life activity tests. Methods: The study involved 15 patients diagnosed with osteoarthritis of the knee and hip joints, aged 62 to 72, mean age 68 ± 3,24 year. Among the respondents, 60% are women and 40% are men. In the study group, 46.7% of people were diagnosed with osteoarthritis of the knee, and 53.3% with osteoarthritis of the hip. The disturbed structures and their functions as well as disturbed activities in everyday life were described using the ICF classification. Activity tests developed by the Polish Chamber of Physiotherapy were used to assess the degree of limitations in everyday activity. Muscle strength was assessed using the Lovet scale, and pain was assessed using the VAS. The range of motion was measured with the goniometer. Results: In the study group from 1 to 4 disturbed structures were observed. The disturbed structures are: 46.6% of people hip joint(s75001); 80% of people thigh muscles(s75002), 46.6% knee joint(s75011), 20% calf muscles(s75012), 6.7% of people pelvic muscles(s7402). In all the subjects disturbances in the body functions — the mobility of a single joint (b7100) were observed, in 73.3% of the subjects — disturbances in the strength of individual muscles (b7300) were observed. The observations regarding the disturbed structures and their functions were supported by tests of muscle strength: gluteus, quadriceps and gastrocnemius muscles. The strength of these muscles was weakened — level 4 or 4.5 on the Lovet Scale. The mean range of knee joint flexion was — 117.9 degrees, in 4 subjects the lack of full extension in the joint was observed. The average results obtained for mobility of hip joint are: 102.23 degrees of flexion, 26.74 degrees of abduction, 20 degrees of adduction, 31.46 degrees of external rotation and 24.67 degrees of internal rotation. In the study group, from 1 to 3 disturbed activities of daily life were observed. The most frequently observed disturbances in the activity of d4551 climbing — in 66.7% of people. 40% of respondents had a problem with the activity of d4103 — moving from sitting to standing, and 26.7% with the activity d4101 — assuming a squatting position and d4500 — walking short distances. The smallest number of respondents (13.3%) had disturbances in the activity of d4102 — kneeling position, d4104 — long-term standing and d4501 — walking long distances. From 1 to 3 activity tests, adequate to the described activity, were performed in the subjects. In the squat position test, the mean distance from the trochanter to the lateral ankle in the examined patients was 57.75 cm, pain in VAS scale — 5.5. In the kneeling position test, the time after which pain appeared in this position was assessed — on average it was 3.75 minutes, Vas — 6. Transition from sitting to standing took the subjects an average of 5.85 seconds, Vas — 4. During the test, 66.7% of subjects needed to use the support on the chair arm to stand up. In the Up & Go test, the mean time with diseased side turnover was 19.29 seconds. 50% of respondents had to use a crutch during the test. The 6-minute walk test was used to evaluate long-distance walking. The average distance covered in the given time was 370m. In the staircase walking test, the time to ascend 1/2 floor was assessed. In the study group it took an average of 16.48 seconds, pain on the Vas scale 4.33 and with average fatigue 4.85. All subjects used the support on the railing while climbing. Conclusion: 1. The study showed a decrease in the functional state caused by the restriction of the range of motion in the hip and knee joints and the weakening of the gluteal, quadriceps and gastrocnemius muscles. 2. The most disturbed activities are climbing stairs, moving from a sitting position to a standing position, taking a squatting position and walking short distances.

LP21 — Neurodegenerative C. Elegans Strains Show Activity Improvement when Bacterial Folate Synthesis is Dampened

Giulia Zavagno^1,2, Michael Fasseas², Adelaide Raimundo², Chris Saunter², David Weinkove^1,2 (1. Durham University — Durham (United Kingdom), 2. Magnitude Biosciences — Sedgefield (United Kingdom))

Background: Many treatments for Neurodegenerative Diseases (NDs) such as Alzheimer’s and Parkinson’s have failed in clinical trials despite promising results in mouse models. The strongest predictor for ND is age, and understanding the ageing body is important to understand how the disease progresses and how the patient responds to treatment. Gut microbiome dysbiosis is also seen with age and in ND patients, and recent research suggests the gut may be responsible for the initiation of disease (Chandra et al. 2017). Therefore, therapies targeted at the microbiome may alleviate ND symptoms but may also act to protect from disease progression. Research into NDs and the microbiome in an ageing model can be challenging. Our group uses the nematode C. elegans to act as a fast readout of ageing and a system to study how bacterial metabolism and drugs affect worm health. Previously, we have shown that inhibiting bacterial folate synthesis in their foodstuff, live E. coli using sulfamethoxazole (SMX), increases worm lifespan (Virk et al. 2012). We have developed an automated monitoring system which tracks worm movement and speed. This technology allows us to test drugs which slow ageing quantitatively in C. elegans models of neurodegeneration, including possible microbiome-targeting drugs like SMX. There have been several transgenic strains created where human proteins such as AB peptide and polyQ, which are found in ND, are overexpressed in specific C. elegans tissues. In these models, the overexpressed protein disrupts cell proteostasis and disrupts function, as seen by changes in movement. Objectives: — Optimise automated monitoring system to detect movement in C. elegans models where disruption of cell proteostasis is induced; — Test the effects of reduced bacterial folate synthesis on C. elegans with aggregating protein expression. Methods: Larval stage 4 worms expressing polyQ and ABeta were placed on OP50 E. coli lawns and monitored non-invasively for seven days. Movement and speed were calculated as they matured into adulthood and began midlife decline. SMX was added to the agar before the growth of the E. coli bacterial lawn. Results: The folate-dampening drug SMX slows decline in wild type worms but has an even greater effect in worms overexpressing polyQ and ABeta peptides. This result suggests that worms suffering from loss of cell proteostasis are particularly sensitive to reducing folate-dependent bacterial toxicity. Conclusion: E. coli can accelerate ageing in C. elegans, and reduction of this activity using SMX improves mobility outcomes especially in ND models. The molecular mechanism by which bacteria accelerates ageing may not be through the action of folates directly but rather require folate-dependent metabolism in E. coli. Research using the C. elegans — bacteria model has yielded information on how E. coli curli protein increases alpha-synuclein aggregation (Chen et al. 2016) and how Bacillus subtilis instead of E. coli can improve such aggregation (Goya et al. 2020). Therefore, C. elegans research may reveal targets in the microbiome, which is a promising route for therapies to delay ageing. C. elegans can also be used as a platform to drug candidates. Our data suggests that slowing ageing by targeting the microbiome may also slow the onset of ND too. References: Chandra et al. (2017) α-Synuclein in gut endocrine cells and its implications for Parkinson’s disease. JCI Insight. 2(12); e92295. Chen et al. (2016) Exposure to the Functional Bacterial Amyloid Protein Curli Enhances Alpha-Synuclein Aggregation in Aged Fischer 344 Rats and Caenorhabditis elegans. Sci Rep. 6; 34477. Goya et al. (2020) Probiotic Bacillus subtilis Protects against α-Synuclein Aggregation in C. elegans. Cell Rep. 30(2); 367–380. Virk et al. (2012) Excessive folate synthesis limits lifespan in the C. elegans: E. coli aging model. BMC Biology. 10(1); 67. Conflict of Interest: GZ recieves funding from Magnitude Biosciences Ltd. DW is a shareholder of Magnitude Biosciences Ltd. MF, AR and CS are employers or shareholders in Magnitude Biosciences Ltd.

Mitochondrial Metabolism and Aging

P32 — Metabolomics-Based Biomarkers for Aging in Chinese Adults

Yiming Pan¹ (1. Department Of Geriatrics, Xuanwu Hospital, Capital Medical University — Beijing (China))

Background: Aging is an inevitable process associated with impairments in multiple organ systems, which increases the risk of comorbidity and disability, and reduces the health-span. Metabolomics is a powerful tool in aging research, which can reflect the characteristics of aging at the level of terminal metabolism, and may contribute to the exploration of aging mechanisms and the formulation of anti-aging strategies. Objective: To identify possible biomarkers and pathways associated with aging using untargeted metabolomics methods. Methods: Liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics profiling was performed on serum samples from 32 older adults and 32 sex-matched young controls. Results: Metabolite profiling could distinguish the two groups. Among the 349 metabolites identified, 80 — including lysophospholipids whose levels gradually decline — are possible candidate aging biomarkers. Valine, leucine and isoleucine degradation and biosynthesis were important pathways in aging, with reduced levels of L-isoleucine and L-leucine observed in older adults. Conclusions: We preliminarily revealed the metabolite changes associated with aging in Chinese adults. Decreases in mitochondrial membrane-related lysophospholipids and dysfunction of branched-chain amino acid metabolism were determined to be the characteristics and promising research targets for aging.

P34 — Functional Transcriptomic Analysis Of Centenarians’ Offspring Reveals a Specific Genetic Footprint That May Explain that They are Less Frail Than Age-Matched Non-Centenarians’ Offspring

Consuelo Borras¹, Marta Ingles², Jose Vina¹ (1. Freshage Research Group, Department Of Physiology, Faculty Of Medicine, University Of Valencia, Centro De Investigación Biomédica En Red Fragilidad Y Envejecimiento Saludable-Instituto De Salud Carlos Iii (ciberfes-Isciii), Incliva- Valencia (Spain), 2. Freshage Research Group, Department Of Physiotherapy, Faculty Of Physiotherapy, University Of Valencia, Ciberfes-Isciii, Incliva — Valencia (Spain))

Background: Centenarians exhibit extreme longevity and compression of morbidity. We showed that centenarians display a unique genetic signature, in terms of mRNA and miRNA profile, which is different from that found in septuagenarians. Centenarians’ offspring seem to inherit centenarians’ compression of morbidity, as measured by lower rates of age-related pathologies. Objective: To ascertain whether centenarians’ offspring are less frail and whether they are endowed with a “genetic footprint”, which might explain their increased vigor and decreased frailty. Design: Case-control study, matched 1:1 for gender, age ±5 years, and place of birth and residence. Setting: Individuals born and usual residents in La Ribera Health Department of Valencian Community (Spain). The study was conducted between March 2015 and February 2017. Subjects: Cases must have a living parent aged 97 years or older, aged 65–80 years, community-dwelling, not suffering from a terminal illness, or less than 6 months of life expectancy. Controls had to meet the same criteria as cases except for the age of death of their parents (not older than 89 years). Centenarians were individuals 97 years or older. Methods: Frailty phenotype was determined by Fried’s Criteria. We collected plasma and peripheral blood mononuclear cells from 63 centenarians, 88 centenarians’ offspring, and 88 non-centenarians’ offspring. miRNA expression and mRNA profiles were performed by the GeneChip miRNA 4.0 Array (Thermo Fisher Scientific) and GeneChip Clariom S Human Array (Thermo Fisher Scientific), respectively. Results: We found a lower incidence of frailty among centenarians’ offspring when compared to their contemporaries’ non- centenarians’ offspring (p <0.01). Both miRNA and mRNA expression patterns in centenarians’ offspring were more like those of centenarians than those of non- centenarians’ offspring (p <0.01). Conclusions: Centenarians’ offspring are less frail than age-matched non- centenarians’ offspring and that this may be explained by their unique genetic endowment.

P35 — BCL-XL MICE: Facing Frailty During Aging

Aurora Román-Domínguez¹, Cristina Mas-Bargues¹, Consuelo Borrás¹ (1. University Of Valencia — Valencia (Spain))

Background: Previous studies performed by our team revealed that centenarians overexpress the anti-apoptotic protein BCL-XL in peripheral blood mononuclear cells (PBMCs), exhibiting decreased markers of apoptosis and enhanced mitochondrial function, along with preserved lymphocyte function. Further studies with C. elegans showed that a gain of function mutation in the BCL-XL orthologue ced-9 (n1959) increased longevity compared to wild type. Taking together, these data suggest a potential role for BCL-XL in successful aging. For this reason, we purposed a murine model which overexpresses human BCL-XL in lymphoid tissues to elucidate the molecular mechanisms that could be responsible for the beneficial effect of BCL-XL in aging. Objectives: The major goal of this study is to characterize the lckpr-bcl-XL transgenic mice in terms of physical performance, frailty, and mitochondrial respiratory function. Methods: First, we obtained a C57BL/6J mice cohort of wild type (WT) and lckpr-bcl-XL transgenic mice (TG) that express a human BCL2L1 cDNA sequence under the control of a mouse Lck promoter. Animals were housed in quiet facilities under a 12 h light/dark cycle (light on at 07:00 a.m.) and maintained in temperature-controlled (23 ± 1°C) rooms with ad libitum access to food and water for the whole duration of the experiment. At the beginning of the study, to characterize BCL-XL protein expression in our murine model, a total of 20 mice from both sexes were sacrificed to obtain several organs and tissues, including the thymus and spleen. For the experimental design, we classified our mice cohort into 4 age groups: 60–180 days-old (young), 240–420 days-old (adult), 540–660 days-old (old), and over 720 days-old (very old). When mice reached each group of age, samples of urine, feces, and blood were collected, and physical tests were performed to assess the frailty score. Grip strength, rotarod, and incremental treadmill tests were performed along with bodyweight measurement. At the end of the tests, mice were sacrificed to obtain freshly isolated spleen T cells by the EasySep™ Mouse T Cell Isolation Kit to perform cellular assays. Mitochondrial function in fresh T cells was assessed with the Cell Mito Stress test by the XFe96 extracellular flux analyser. For the statistical analysis, t-test or ANOVA were performed if the assumptions were met. Otherwise, the Wilcoxon test or Kruskal-Wallis rank-sum test was performed as non-parametric alternatives. The alpha level was set at 5% as a criterion for the acceptance of the null hypothesis. For the analysis of the mitochondrial function, z-score normalization was performed to reduce the variability between plates with the same experimental groups. Results: Transgenic mice showed increased levels of BCL-XL in the spleen and thymus when compared to WT mice, as expected. In addition, transgenic mice showed improved grip strength, increased latency time during the rotarod test, and lower running time during the incremental test, along with a lower increase in bodyweight during aging. The frailty score was assessed for the mice cohort at old age, showing a lower score in transgenic mice compared with wild type, but the result was not statistically different. Regarding mitochondrial respiration in fresh T cells, cells from young and adult mice recorded lower levels of basal respiration, proton leak, and non-mitochondrial respiration along with higher coupling efficiency compared to WT. However, these differences were lost at advanced ages. Conclusion and discussion: Although the role of BCL-XL in the physiology of aging remains unclear, our results suggest its involvement in bodyweight regulation, explosive strength, and frailty in vivo. However, the molecular mechanisms of these events are still unknown. In the mitochondria, BCL-XL influences the morphology and affects cellular energy metabolism. In this regard, our results suggest a major function of BCL-XL in the proton gradient efficiency along the electron transport chain, that is lost with aging. Although many questions remain, the transgenic mice reveal new potential functions of BCL-XL in mitochondrial homeostasis, cellular response to stress, and overall body physiology.

P36 — Genistein in the Control of AD Progression. From Molecular Mechanisms to Clinical Improvement: The Genial Clinical Trial (NCT01982578)

Jose Viña¹, Consuelo Borras¹ (1. Freshage Research Group, Department Of Physiology, Faculty Of Medicine, University Of Valencia, And Ciberfes, Insitute Of Health Research-Incliva — València (Spain))

Background: Alzheimer’s disease has devastating effects on the adult population and caregivers. It is an age-associated disease. One of the most important risk factors is the presence of the ApoE 4 allele. New drugs have been sought to delay neurodegeneration, but we have concentrated on nutritional approaches to the improvement of patients with neurodegeneration. Some ten years ago it was reported (1) that bexarotene can delay the onset of Alzheimer’s disease in mouse models. By analogies between the bexarotene receptor and the genistein receptor, we thought that genistein, a component of soy, could have an effect analogous to bexarotene in terms of improving cognition, brain biochemistry, and brain structure in mouse models of Alzheimer’s. We observed that genistein performed as well as bexarotene, if not better, in treating Alzheimer’s disease in mice (2). The next step, of course, was extrapolation to Alzheimer’s disease in humans. We first did a pilot test where it was shown that the administration of genistein (60 mg, twice a day) was more effective than placebo in delaying the onset of dementia in a small number of minimal cognitive impairment patients. Objective: To perform to a clinical trial with genistein do determine its effectiveness in delaying the onset of Alzheimer’s dementia in minimal cognitive impairment patients. Methods: Before and after a twelve-month intervention, we determined several parameters of cognition, among which are the most used in clinical practice like, for example, in the mini-mental state examination. The trial was double-blind, placebo controlled, randomized, bi-center study (carried out at the General Hospital of Valencia, coordinated in that hospital by Dr. Escudero and in the Hospital Universitario La Fe de Valencia, coordinated in that hospital by Dr. Vaquero). Results: Cognitively impaired individuals who took genistein after twelve months were significantly better in terms of cognition than those who took placebo. Of the twelve tests we analyzed, two of them came out directly significant. And in all the rest, it was seen that the improvement was greater in the case of genistein than in the controls, although the individual result in each case was not significant. This had happened before in another analysis with aducanumab (3) and the authors developed a “composite score of cognitive impairment”. Similarly, we did a global test based on each of the individual tests of cognitive function, and it was shown that genistein is significantly better than placebo in delaying the onset of cognitive deficits for one year. The values are statistically significant. Conclusion: This study shows that a chemically defined component of soy, genistein, may have a role in therapeutics to delay the onset of Alzheimer’s dementia in patients with mild cognitive impairment, the improvement is similar or even better to that reached with other recently approved compounds like aducanumab.

P37 — BCL-XL Overexpression Protects T Cells During Aging: Balancing Apoptosis and Autophagy

Cristina Mas-Bargues¹, Aurora Román-Domínguez¹, Jorge Sanz-Ros¹, Mar Dromant¹, Nekane Romero-García¹, Jose Viña¹, Consuelo Borrás¹ (1. University Of Valencia — Valencia (Spain))

Background: The world’s centenarian population has remarkably increased in the last decades. Centenarians exhibit low incidence rates of common age-related disorders. Therefore, they have been proposed as a model of “successful aging”. Many studies have been carried out to determine whether centenarians live longer and are healthier than their contemporaries due to genetic, epigenetic, or environmental factors. Since centenarians are exposed to the same basic environmental conditions as their contemporaries who experience an “ordinary aging”, it is deduced that genetic factors may play a critical role in their longevity. For this reason, we obtained peripheral blood mononuclear cells (PBMCs) from young people, octogenarians and centenarians, and directed our transcriptomic analysis towards the identification of mitochondria-related genes which are specifically upregulated in centenarians, and we converged in one: BCL-xL. Indeed, we found that centenarians overexpress BCL-xL and that C. elegans with a gain of function of the BCL-xL ortholog live longer than controls. Beyond its well-described role as an inhibitor of mitochondrial apoptosis by preventing cytochrome c release, BCL-xL has also been related to the regulation of mitochondrial energetics by stabilizing the inner membrane potential. Moreover, recent findings suggest a mechanism of action: it binds to respiratory complex V and in this way, BCL-xL can regulate the crosstalk between apoptosis, autophagy, and senescence, thus promoting cell survival. Objective: The main aim of this study is to explore the role of mitochondria in the development of energy collapse in aging. We used C57BL6/J mice that overexpress the human BCL-xL gene in spleen and thymus (similarly as in centenarians), analyzed longitudinally at different ages: 2–4, 8–10, 18–20, and >24 months old. We performed in vitro molecular studies to evaluate oxidative stress, apoptosis, senescence, and autophagy-related parameters in splenic T cells from WT and BCL-xL transgenic mice. Methods: All the animals used for this project were housed in the animal house facility of the Central Medical Research Unit (UCIM) of the Faculty of Medicine, under conditions of temperature (23±1°C), relative humidity (60%), and constant light/dark cycles (12h/12h), and fed with a standard laboratory diet and water ad libitum. We used C57BL6/J mice as wild-type (WT) and heterozygous Lckpr-bcl-XLtransgenic (TG) mice with C57BL6/J genetic background. These latter animals overexpress the BCL-xL in T cells, spleen, and thymus. For the experimental design, we classified our mice cohort into 4 age groups: 2–4 months-old (young), 8–10 months-old (adult), 18–20 months-old (old), and over 24 months-old (very old). At each group of age, some animals were sacrificed and spleens were immediately isolated and placed in PBS. Single-cell splenocyte suspensions were then processed with the STEM CELL EasySep mouse T cell isolation kit (Stem Cell Technologies). Splenic T cells were analyzed for senescence (FDG), apoptosis (Annexin V), autophagy (LC3-II), and oxidative stress-related parameters (TMRM and MitoPY1) by flow cytometry. Results: T cell senescence increases with age both in WT and TG mice, without significant differences between them. In opposition, T cell apoptosis decreases with age in WT and TG mice, although only old and very old TG mice keep significantly higher levels of apoptosis compared to their WT counterparts. Moreover, in vitro cultures of T cells submitted to survival tests (Control, Dexamethasone, or Starvation) revealed that only young and adult TG T cells have lower levels of apoptosis compared to WT T cells. In addition, in vitro cultures of old and very old T cells showed that only TG T cells were able to upregulate autophagy in response to starvation stress in comparison to their WT counterparts. Finally, mitochondrial membrane potential and mitochondrial peroxides level remain constant through aging, without significant differences between WT and TG mice. Conclusion and discussion: Although the role of BCL-XL in the physiology of aging remains unclear, our results suggest its involvement in apoptosis and autophagy regulation. Our results indicate that BCL-xL antiapoptotic properties protect T cells from external insults until adulthood. On the other hand, BCL-xL protects T cells from internal damage in late life, where it also upregulates macroautophagy to promote cell survival. Taken together, these results suggest an important role for BCL-xL in T cell survival during aging. Although BCL-xL did not affect the mitochondrial membrane potential and the mitochondrial peroxides level of T cells, it could be interesting to analyze whether BCL-xL modulates mitochondrial bioenergetics. Future studies will also assess whether the effect of BCL-xL in T cells influences other tissues and ameliorates frailty.

P38 — Reconstruction of Functional Human Epidermis Equivalent Containing 5 %IPS-Derived Keratinocytes Treated with Mitochondrial Stimulating Plant Extracts

Anne-Laure Bulteau¹, Mariele Moreau¹, Christophe Capallere¹, Christelle Plaza¹, Carine Nizard¹ (1. Lvmh Research — St. Jean De Braye (France))

Background: Reconstructed human epidermis equivalents (RHE) have been developed as a clinical skin substitute and as the replacement for animal testing in both research and industry. KiPS, or keratinocytes derived from induced pluripotent stem cells (iPSCs) are frequently used to generate RHE. Objectives: Reconstruction of RHE using Kips from different age donors and study mitochondrial function between iPS and KiPS. Methods: We used seahorse technology to explore mitondrial function in iPS and KiPS and in RHE. Results: In this study, we focus on the mitochondrial performance of the KiPS derived from iPSCs obtained from two donors. We found that the KiPS derived from the older donor have more defective mitochondria. Treatment of these KiPS with a plant extract enriched in compounds known to protect mitochondria improved mitochondrial respiration and rendered them fully competent to derive high-quality RHE. Conclusion: Overall, our results suggest that improving mitochondrial function in KiPS is one of the key aspects to obtain a functional RHE and that our plant extracts can improve in this process.

P39 — Inhibition of DE Novo Ceramide Synthesis Promotes Skeletal Muscle Hypertrophy in Young Mice but Does Not Prevent Sarcopenia in Old Mice

Benjamin Lair¹, Geneviève Tavernier¹, Virginie Bourlier¹, Aline Mairal¹, Camille Bergoglio¹, Diane Beuzelin¹, Deborah Carper¹, Claire Laurens¹, Cedric Moro¹ (1. Inserm, Umr1297, Institute Of Metabolic And Cardiovascular Diseases, Paul Sabatier University- Toulouse (France))

Background: Sarcopenia is an age-related condition that is characterized by progressive loss of muscle mass and force. Muscle mass maintenance partly relies on the balance between protein synthesis and proteolysis. Several studies indicate that sarcopenia is characterized by a reduced protein synthesis rate to anabolic stimuli that include feeding and exercise leading to muscle mass loss overtime. Some studies suggested a possible accumulation of bioactive lipids such as ceramides in aged skeletal muscle. Considering that ceramides have been shown to inhibit anabolic signalling, we here hypothesized that ceramide could contribute to anabolic resistance and muscle mass loss during aging. Objectives: The goal of this study was to investigate the role of ceramides and other specific sphingolipids in the pathogenesis of sarcopenia. We aimed to determine if a pharmacological inhibition of de novo ceramides synthesis exerted a geroprotective effect on muscle mass loss. Methods: To determine the role of ceramides in sarcopenia, we treated adult (7 months) and aged (23 months) mice for 6 weeks with myriocin (an inhibitor of de novo ceramide synthesis) using subcutaneous osmotic pumps. Muscle (Tibialis anterior) and liver sphingolipids were analyzed by LC-MS. Cross-sectional area (CSA) of gastrocnemius muscle fibers were measured through automated segmentation. In parallel, cell surface was measured in C2C12 myotubes treated with ceramides and myriocin. Results: As expected, old mice exhibited reduced grip strength, hindlimb muscle weight and gastrocnemius fiber CSA compared to younger counterparts. Muscle ceramide content was elevated in aged mice mainly due to C24:1 ceramide accumulation. In both groups, myriocin treatment remodelled sphingolipid species distribution in muscle and liver. Surprisingly, myriocin treatment significantly increased the CSA of type IIa and IIb/IIx fibers in young mice. A similar effect was observed in vitro in cultured C2C12 myotubes. However, no effect was observed in aged mice. The underlying molecular mechanisms are currently being investigated. Discussion: Altogether, these data demonstrate a positive effect of de novo ceramides synthesis inhibition both in vitro on myotube size and in vivo and on adult mouse muscle fiber CSA. However, no preventive effect on sarcopenia was observed in aged mice.

P40 — Models of Mechanosensitive Amyloid Channels Involved in Mitochondria Swelling

H. Robert Guy¹ (1. Amyloid Research Consultants — Cochiti Lake, Nm (United States))

Amyloid-forming peptides and proteins underlie or are involved in diseases prevalent among the elderly including Alzheimer’s (Amyloid β or Aβ), Parkinson’s (α-Synuclein or α-Syn), Type 2 diabetes (Amylin or IAPP), and some cancers (γ-Synuclein). These devastating diseases kill millions of people and cost billions of dollars annually. Studies of Aβ and α-Syn fibrils have produced no effective treatments or drugs, perhaps because much smaller oligomers are the toxic culprits. These amyloid oligomers interact with numerous membranes that include the inner membrane of mitochondria. These interactions appear to alter the mitochondria’s morphology and viability. Although it has long been known that mitochondrial swelling and disfunction involves a permeability transition pore (PTP), the identity of all proteins forming the PTP complex have still not been determined. One suggestion has been that amyloids are involved. All the amyloids mentioned above form transmembrane channels of various sizes. Antibody studies suggest that they all share a common structural feature with α hemolysin, which forms a transmembrane antiparallel β-barrel channel. The first goal of our research is to develop structural models of these oligomers as they form in solution, interact with and span membranes, cluster to form channels between the oligomers, morph to form larger concentric β barrel channels, and expand when the membrane is stretched. Our models suggest experimental tests and ways to stabilize specific structures so their 3-D structures can be solved experimentally. Perhaps most important, they suggest multiple targets and ways to prevent, treat, and possibly cure these devastating diseases. Utilizing well-established β-barrel theory and protein modeling methods, techniques, constraints, and criteria, we have developed β-barrel models of Aβ and Synuclein oligomers, annular and cylindrical protofibrils, lipoproteins, and transmembrane channels. We favor models that maximize energetically favorable interactions: hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains. Residues with polar side-chains and/or that have a high propensity for turn or coil secondary structure are favored for connecting loops. Also residues that are identical among distantly related homologous proteins (e.g., α-Syn and β-Syn families) cluster in the interior or at functional sites whereas residues that are hypervariable and/or where indels occur among families are located primarily on protein surfaces. The models have few monomeric conformations, which requires that their subunits be symmetrically related. Our models are developed to be consistent with experimental results; e.g., microscopy studies of the sizes and shapes of oligomers, annular protofibrils, lipoproteins, and membrane channel, biochemical and biophysical studies, NMR distances, molecular weights, secondary structures, toxicity, single channel conductances, and effects of oxidation and heavy ion binding. We have developed atomic scale models of some of these assemblies and analyzed the stabilities of some using multiple types of molecular dynamic simulations. For most models each monomer contains multiple β-strands: Aβ42 has three or four and αSyn has seven or eight. Most of our models antiparallel β barrels. The most hydrophobic β-strands form the core β-barrel of our soluble oligomers and the exterior β-barrel of our transmembrane models. The other strands can be either buried or exposed to water depending upon the model. Aβ42 assemblies are described here. The size of Aβ42 oligomers vary: they can be tetramers, hexamers, octamers, dodecamers, hexadecamers, and octadecamers. These oligomers cluster to form rings of various sizes and shapes in beaded annular protofibrils (bAPFs). With time bAPFs morph into smooth annular protofibrils (sAPFs), which we model as a single set of concentric β-barrels with the same number of monomers as their bAPF precursors. The oligomers can also interact with membranes to form transmembrane oligomers (TMOs). Often only the hydrophobic core β-barrel resides in the transmembrane region. These TMOs can cluster to form assemblies of two to six TMOs. For large assembles a pore can form through the center of these clusters. With time these TMO cluster can merge to form larger pores through the center of a concentric β-barrel assembly. The hydrophobic β-strands comprise the exterior β-barrel while more polar strands comprise the pore lining. When the membrane is stretched, the tilts of the β-strands relative the central axis may increase. This change shortens the hydrophobic exterior of the assembly and increases its diameter, including that of the pore. A similar process occurs in mechanosensitive channels of known structures, such as MSCL. In fact, each step in the formation of the channels: (insertion of oligomers into the membrane, aggregation of TMO with pores in their centers, morphing of TMO clusters to concentric β-barrels, and increase tilting of the β-strands to form shorter and wider channels) should be facilitated by membrane stretching. Similar processes could occur for α-Syn channels. We thus conclude (hypothesize) that these amyloids may be involved in mitochondrial disfunction associated with numerous diseases.

P41 — The Obage Study: A Prospective Case-Control Study Embedded in a Prospective Birth Cohort to Identify Systemic, Cellular, and Molecular Biomarkers of Obesity-Induced Accelerated Aging in 30-Years Old Chilean Adults

Maria Paulina Correa¹, Raquel Burrows¹, Cecilia Albala¹, Felipe Salech², Guillermo Sanhueza³, Gonzalez-Billault Christian⁴ (1. Inta, Universidad De Chile — Santiago (Chile), 2. Faculty Of Medicne, Universidad De Chile — Santiago (Chile), 3. Faculty Of Social Science, Universidad De Chile — Santiago (Chile), 4. Faculty Of Science And Faculty Of Medicine, Universidad De Chile; Geroscience Center For Brain Health And Metabolism (gero), Chile — Santiago (Chile))

Background: Aging is characterized by a progressive loss of capacities linked to fundamental alterations and damage in multiple cellular and molecular pathways. As such, most non-communicable diseases (NCDs) have aging as their primary risk factor. Overweight and obesity have also become relevant contributing factors challenging the Chilean population’s health status in the last decades. It has been suggested that obesity accelerates the onset of metabolic imbalances, decreases lifespan, and impacts cellular and molecular processes like aging. Thereby, obesity might accelerate the pace of aging, affecting all aspects of physiology and, thus, shortening health span and life span. Objective: We recently started a study protocol for Obesity-induced accelerated Aging (ObAGE) that aims to explore how exposure to obesity in critical developmental stages disrupt homeostatic resilience mechanisms that preserve physiological integrity and induce the early expression of aging phenotypes. Also, we aim to determine whether exposure to early psychosocial adversity influences vulnerability to obesity as a risk factor for accelerated aging. Methods: The current study will capitalize on a prospective epidemiological cohort (Santiago Longitudinal Study, SLS) of ≈1000 Chileans at 30–31y, 50% females, of low-to-middle socioeconomic status, who participated in research related to nutrition and development since birth with follow-up at 1-5-10-12-14-16-21-23y. In addition, cardiometabolic evaluations were included at 17 and 23y, with a third assessment wave starting in late 2021. Previous work indicates that at 23y, 25% of participants were obese, 14% had metabolic syndrome, 47% had insulin resistance, 28% had non-alcoholic fatty liver disease (NAFLD), and 27% had low-grade systemic inflammation. Also, a poorer infant psychosocial environment in these participants was associated with a higher Body-Mass Index (BMI), blood pressure, triglycerides, total cholesterol, high-sensitivity C-reactive protein, and greater prevalence of insulin resistance metabolic syndrome in adolescence and early adulthood. With these antecedents, we are conducting a multiple events case-control study embedded in our prospective cohort. To be eligible for the ObAGE study, participants must have complete data in all assessments waves and fall into one of the following categories of lifetime BMI trajectory: (1) participants always having a BMI in the healthy range; (2) participants with obesity starting in adolescence and remained obese into adulthood; (3) participants who were obese in early childhood and remained obese into adulthood. To evaluate whether obesity is contributing to an increased aging pace, we will use a multi-layer approach that includes the following measurements and procedures: anthropometric assessment (weight, height, BMI, WHtR, WSR); a dual-energy X-ray absorptiometry scan for body composition will determine total and percent body fat mass, total and percent lean mass, bone mineral density and bone mineral content in the total body and specific regions; an abdominal ultrasound will be performed for diagnosis of NAFLD, and alanine and aspartate aminotransferase will be determined (enzymatic method); stool samples will be collected, and the ribosomal RNA 16S gene will be sequenced (Illumina) to characterize the gut microbiome; a panel of age-related biomarkers will be determined in biological samples: IGF-1, IGF-2, OSM, myostatin, Fgf21, Gdf15, Gdf11, Apelin, Musclin, Sparc, Irisin, IL-2, IL-6, IL-10, TNFα, CRP y SAP; for the first time in Chile, we will address age-related epigenetic changes that could be passed on to offspring, seeking to understand better the mid-and long-term consequences of obesity for human health, using the epigenetic clock developed by the Horvath’s group. After characterizing the three groups, we will conduct a controlled physical challenge to evidence poorer physical resilience in the obese groups compared to the normal-weight subjects. Results: We expect to go beyond classical epidemiological approaches to understand better how exposure to obesity in childhood, adolescence, and early adulthood disrupts homeostatic resilience mechanisms that prevent the early expression of aging phenotypes. Secondly, we aim to contribute to a deep comprehension of the role of social position in the vulnerability to obesity-induced accelerated aging. Because SLS participants are still young, our research setting combined with advanced scientific techniques may identify individuals or groups at risk of early-onset cardiometabolic problems, Alzheimer’s disease, osteoarthritis, and other age-related health issues. In the long run, we expect to create the basis for future studies on the transgenerational effects of obesity-induced accelerated aging by enrolling children born to SLS participants. Conclusion: Results collected from this study may pave the way to address the contribution of obesity to aging through lifespan from cells to systems and might be instrumental to developing interventions to improve healthspan in the Chilean population. Funding: ACT #210006, FONDAP #15150012; FONDECYT #1210283. Competing interests: None declared.

P43 — Oxidative Metabolism Response to High Fat Diet in a Mouse Model of Neurodegenerative Disease

Camille Champigny^1,2, Djamaa Atamena², Marlène Botella², Sébastien Bullich², Bruno Guiard², Marie-Christine Miquel², Corentin Coustham^2,3, Pascale Belenguer², Noélie Davezac² (1. CHU Toulouse — Toulouse (France), 2. CRCA-CBI — Toulouse (France), 3ISAE-SUPAERO — Toulouse (France))

The aging of population is global phenomenon with the number of people aged 80 and over is expected to triple, from 143 million in 2019 to 426 million in 2050, according to the World Health Organization. It is explained by increasing life expectancy and improved survival at younger ages. The World Health Organization encourages prevention of the decline in the physical and mental capacities and to anticipate the loss of autonomy. Because, some cases of older people develop pathological and irreversible aging compared to others, who are frail associated with reversible status. The progressive decline of physiological functions is characterized by hallmarks including mitochondrial dysfunctions. Indeed, mitochondria plays an important role in metabolism and more precisely in oxidative metabolism. When they are altered, their dysfunctions may be involved in various pathologies and in particular in neurodegenerative diseases. Furthermore, lifestyle, such as nutrition, can have a positive or negative effect on the maintenance of metabolism. Mitochondria is central in the energy metabolism by producing ATP through oxidative phosphorylation. It also participates in the Reactive Oxygen Species (ROS) production by the mitochondrial respiratory chain. In the cell, ROS are detoxified by antioxidant defences. This mechanism allows to maintain the redox steady state. Mitochondria appear as a major characteristic of functional capacities decline, in particular by ROS accumulation and the associated damage. We previously showed that oxidative metabolism is a key player is an optic neuropathy: Dominant Optic Atrophy associated to OPA1 gene mutations (Millet et al., 2016). OPA1 is a mitochondrial protein, essential in the fusion process. Up to 20% of patients develop extraocular neurological complications such as deafness, parkinsonism syndrome, ataxia. We demonstrated that aging has an impact on the oxidative metabolism machinery (Daloyau et al., 2018). In this work, we used this mouse model of the pathology to study the impact of high fat diet (HFD) on oxidative metabolism and identify biomarkers of redox state imbalance. We studied cortices from WT and OPA+/- mice fed 3 months with HFD. The antioxidant machinery was analysed as well as molecular signature of ROS production. The last results will be presented. Daloyau, M., Millet, A., Miquel, M.-C., Mils, V., Wissinger, B., Belenguer, P., Davezac, N., 2018. Brains from Aged OPA1+/-(B6;C3-Opa1 329–355del) Mouse Strain Are in a Pro-Oxidative State. React. Oxyg. Species. 10.20455/ros.2018.863. Millet, A.M.C., Bertholet, A.M., Daloyau, M., Reynier, P., Galinier, A., Devin, A., Wissinguer, B., Belenguer, P., Davezac, N., 2016. Loss of functional OPA1 unbalances redox state: implications in dominant optic atrophy pathogenesis. Ann. Clin. Transl. Neurol. 3, 408–421. 10.1002/acn3.305

P44 — Examining Human Fertility as a Fitness Component in the Context of the Antagonistic Pleiotropy Theory of Ageing

Eva Brigos¹, Carlos Morcillo-Suárez¹, Gerard Muntané¹, Arcadi Navarro¹ (1. Department Of Experimental Health Sciences, Institute Of Evolutionary Biology (upf-Csic) — Barcelona (Spain))

Background: The agrarian and industrial revolutions marked the start of a new period in human development with major transitions occurring in nutritional, epidemiological and demographic terms. As a result, modern humans live in conditions that are radically different from those under which we evolved. Maturity, fertility and lifespan are examples of life history traits impacted by mismatches between past adaptations and current environments. As exposed by the antagonistic pleiotropy theory of human ageing (AP; Williams, 1957), genetic variants causing damage to the organism in post-reproductive ages (e.g., associated with late-onset diseases) might present higher frequencies in the population and larger effect sizes as a result of evolutionary trade-offs with fitness-contributing early-onset traits. While this has been demonstrated by our group for the case of complex diseases, an unexplored prediction of AP is that, in modern societies, variants that promote fitness through increased fertility may be, at the same time, predisposing factors for late-onset non-communicable diseases. Objectives: By integrating extensive data on GWAS summary statistics we want to assess the evidence supporting pleiotropy between fertility-related traits and human complex diseases. Specifically, we aim at deciphering the genetic trade-offs, if any, between fertility and both early and late-onset diseases, accounting also for the effect direction in both traits. This will improve our understanding of human evolution and complement global efforts to understand the impact that non-communicable diseases have in human health of modern aged populations. The main objective of this study is to analyse the pleiotropic landscape of fertility as a fundamental fitness component and to understand our findings in the context of the AP theory for human senescence. More specifically: 1) obtain a map of the shared genetic architecture between fertility and human complex diseases; 2) obtain a map of antagonistic and agonistic variants between fertility and early-to-late onset of diseases; 3) screen for signatures of the action of natural selection upon the loci shared between fertility and complex diseases, which might have aroused as a result of selective pressure compromises; and 4) interpret the information gathered above in the context of human ageing patterns, both historical and current. Methods: Throughout this study we use the term fertility to denote the potential for reproduction. As a proxy for fertility we have used three different traits from different genome-wide association studies (GWAS): number of children ever born, number of live births (to a female) and number of children fathered (by a male). In parallel, and after quality-control steps we selected 55 complex diseases from the GWAS Atlas and GWAS Catalog databases. To include the dimension of age in our analysis we assigned an age of disease onset to each of the 55 diseases. We defined three disease onset ages based on 5%, 50% and 95% of cumulative prevalence values. We generated disease prevalence curves by computing cumulative prevalence rates for each age-range in the dataset. We then normalized to 1 the raw counts. We differentiate between positive and negative correlations and pleiotropies (pleiotropic SNPs). Positive when alleles have concordant effects: an increase in fertility is coupled with an increase in disease risk (or vice versa). And negative when alleles have opposite effects. To estimate global genetic correlations (rg) between the fertility traits and the diseases we used the linkage disequilibrium score (LDSC software). By means of these comparisons, we obtained an overall estimation of the sharedness of subjacent genetic architecture between fertility and the set of diseases. We used pleioFDR software to compute the genetic pleiotropic-informed conjunctional false discovery rate (conjFDR) method on GWAS summary statistics. We performed pairwise analysis using the common set of SNPs between the fertility traits and the 55 diseases. We considered shared loci at a threshold conjFDR<0.05. We then used a two-sample Kolmogorov-Smirnov test to assess the dissimilarity in the age-related pattern of the cumulative distributions. Finally, we conducted a preliminary analysis on signatures of natural selection in the human genome using some measures that aid in the interpretation of selective events over our evolutionary history. Result: We show that positive genetic correlations are more common in early-onset diseases. Also, positive and negative pleiotropies have different accumulation curves with positive pleiotropies increasing at earlier ages of disease onset. Likewise, we have identified more positive than negative pleiotropies between fertility and the set of complex diseases. And, when evaluating signals of positive selection our results suggest differential selective pressures acting over positive and negative pleiotropies. Conclusion: Overall, our findings suggest that the role of fertility in shaping modern disease patterns is consistent with the Antagonistic Pleiotropy theory of human ageing.

P46 — Differential Expression of Perilipin Family Proteins in Human Brain During Aging and Alzheimer’s Disease

Maria Conte¹, Valentina Medici², Antonio Chiariello¹, Isidre Ferrer³, Giuseppe Legname⁴, Emanuele Poloni², Antonio Guaita², Claudio Franceschi⁵, Stefano Salvioli¹ (1. Department Of Experimental, Diagnostic And Specialty Medicine (dimes), University Of Bologna — Bologna (Italy), 2. Department Of Neurology And Neuropathology, Golgi-Cenci Foundation — Abbiategrasso (Italy), 3. Department Of Pathology And Experimental Therapeutics, Institute Of Neurosciences, University Of Barcelona — Barcelona (Spain), 4. Laboratory Of Prion Biology, Department Of Neuroscience, Scuola Internazionale Superiore Di Studi Avanzati (sissa) — Trieste (Italy), 5. Institute Of Information Technologies, Mathematics And Mechanics, Lobachevsky University — Nizhniy Novgorod (Russian Federation))

Background: Perilipins (Plins) belong to a family of five evolutionary conserved proteins (Plin1–5) that decorate intracellular lipid droplets and are involved in different cellular process, such as lipid metabolism. Plins are essential for the normal physiology of cells and recent findings indicate that their differential expression is associated to alterations in lipid metabolism leading to several age-related diseases. As an example, several studies demonstrate that in humans high levels of Plin2 are associated with fatty liver diseases, atherosclerosis, obesity, cardiovascular disease, diabetes and sarcopenia. Conversely in mice, the downregulation or knockout of Plin2 prevents or mitigates these pathologies. Objectives: To date little is known about the expression of Plins in human brain and their possible involvement in brain aging and neurodegeneration. Thus, the aim of this study was to characterize the expression levels of Plins in different cerebral areas from subjects of different age, with or without signs of neurodegeneration, such as early tauopathy or Alzheimer’s disease (AD). Methods: We performed real time RT-PCR, western blotting, immunohistochemistry and confocal microscopy analyses in autoptic brain samples of frontal and temporal cortex, cerebellum and hippocampus obtained from subjects ranging from 33 to 104 years of age, with or without histological signs of neurodegenerative pathologies. Moreover, in order to test a possible association between Plins and inflammation, we performed a correlation analysis with IL-6 expression, a pro-inflammatory cytokine involved also in neurodegenerative disorders, and in vitro experiments on primary dermal fibroblast cultures (DFs), now considered a reliable model system to study metabolic abnormalities related to neurodegenerative diseases. In particular, we treated DFs with metformin, a glucose-lowering drug able to modulate lipid metabolism and inflammatory response. Results: Plin2, Plin3 and Plin5, but not Plin1 and Plin4, are expressed at protein level in brain areas. Plin2 appears expressed more in grey matter, particularly in neurons, in all the areas considered, while Plin3 and Plin5 appear more expressed in white matter, Plin3 in particular seems expressed more in astrocytes. At variance with Plin3 and Plin5, Plin2 resulted modulated by age and neurodegeneration, being highly expressed in old subjects and centenarians, and in patients with early tauopathy or AD. Moreover, only Plin2 correlated with the expression of IL-6. In vitro data from DFs treated with Metformin, indicate that expression of Plin2 and IL-6 follow the same trend, suggesting that Plin2 and IL-6 expression may be causally connected. Conclusions: Plins are expressed in human brain with an apparently cell specificity and with different intensities in different areas. Only Plin2 appears modulated with age and neurodegeneration. Interestingly, Plin2 correlates with markers of neuroinflammation. We propose that the accumulation of lipid droplets decorated with Plin2 occurs in neurons during brain aging, and this accumulation may be an early marker and initial step of neuronal stress and possibly of inflammatory and neurodegeneration. Conflict of interest: The authors declare no conflicts.

P47 — Expression Pattern of the Mitokine GDF15 in Human Brain in Healthy Aging and in Alzheimer’s Disease

Antonio Chiariello¹, Maria Conte², Valentina Medici³, Tino Emanuele Poloni³, Antonio Guaita³, Stefano Salvioli¹ (1. Department Of Experimental, Diagnostic And Specialty Medicine (dimes), University Of Bologna, Bologna, Ital — Bologna (Italy), 2.Department Of Experimental, Diagnostic And Specialty Medicine (dimes), University Of Bologna — Bologna (Italy), 3.Department Of Neurology And Neuropathology, Golgi-Cenci Foundation — Abbiategrasso (Italy))

Background: Mitokines are molecules whose expression, both at tissue and circulating levels, rise in response to stress and in particular to mitochondrial dysfunctions. Mitokines are able to induce a compensatory response, even in cells not directly affected by the mitochondrial stress. Among mitokines, Growth Differentiation Factor 15 (GDF15) is induced by a wide range of stresses such as mitochondrial stress, inflammation and endoplasmic reticulum’s stress, and at present is among the most upregulated proteins during aging. Many studies suggest for this protein an anti-inflammatory role, particularly in metabolic regulation, however GDF15 also displays a strong and positive association with different age-related diseases, as well as with overall mortality. GDF15 is regulated at transcriptional levels by different type of transcription factors, such as ATF3, ATF4, CHOP and p53, as well as at post-translational levels. GDF15 is first synthetized as a precursor (pro-GDF15) that dimerizes and is then cleaved in the mature protein. It is though that the effects of GDF15 are mainly mediated by the mature form, but studies speculate that also pro-GDF15 is biologically active. At present the biological function of pro-GDF15 and mature GDF15 is not fully clear and it is still debated whether elevated levels of this mitokine are protective or detrimental, especially in elderly and in age-related diseases. Objectives: Many studies demonstrated that high levels of GDF15 are found in old patients with pathologies such as type 2 diabetes, cardiovascular diseases, sarcopenia and cancer, but to date little is known about the expression of GDF15 in neurodegenerative diseases, such as Alzheimer’s disease. Therefore, the main objective of this study was to characterize the expression pattern of GDF15 in human samples of different brain regions from old subjects, AD patients and centenarians to understand the possible role of this molecule in healthy aging and in neurodegeneration. Furthermore, in the same samples we analyzed and compared the expression of pro-GDF15 and mature GDF15 to evaluate possible differences in GDF15 maturation between AD patients and healthy old subjects. Methods: We performed Real Time RT-PCR and Western Blotting in autopsy human brain samples from 23 subjects in the age range 33–104 years, with or without neurodegeneration. Experiments were carried out using samples from frontal cortex (also gray and white matter separately), hippocampus, cerebellum and dentate nucleus. Results: We analyzed the transcript level of GDF15 and its transcriptional activators (ATF3, ATF4, CHOP and p53) in samples of frontal cortex from 5 AD patients, 3 old subjects without sign of neurodegeneration (O) and 2 centenarians, The transcript level of ATF3, ATF4 and CHOP appeared higher in AD patients and even more in centenarians with respect to O, while p53 transcript level was similar in AD and centenarians, and higher compared to O. Likewise, GDF15 transcript level was higher in AD patients and centenarians with respect to O. As regard pro-GDF15 and mature GDF15 protein expression, we found different expression patterns depending on the brain area considered. In frontal cortex, and similarly in hippocampus, pro-GDF15 expression resulted higher in O and AD patients compared to young adults (YA) and centenarians, who presented the lowest expression of pro-GDF15. The expression of the mature form of GDF15 followed a different pattern, with a lower expression in YA with respect to O, AD patients and centenarians. We then focused on GDF15 protein expression in gray (GM) and white matter (WM) of frontal cortex from O and AD patients. Both Pro-GDF15 and mature GDF15 resulted expressed more in GM than WM, however, no difference was found between the two groups as far as pro-GDF15, while the mature form resulted more expressed in AD patients in WM. In the cerebellum pro-GDF15 protein appeared expressed less in YA compared to O, AD patients and centenarian, conversely the mature form resulted lower in centenarian with respect to all other groups. In the dentate nucleus pro-GDF15 resulted expressed less in AD patients compared to O, and the mature GDF15 had an opposite trend. Conclusions: GDF15 expression in the brain tissue appears to be affected by aging and neurodegeneration, with a brain area-specific pattern. In particular, in each area considered the pro-GDF15 and the mature form followed a different trend. We speculate that in addition to the modulation of the expression of GDF15, its protein processing could play a role in brain aging and neurodegeneration. Conflict of interest: The authors declare no conflicts

LP23 — Circulating Levels of Apelin, GDF-15 and Sarcopenia: Lack of Association in the MAPT Study

Juan Luis Sanchez Sanchez¹, Lingxiao He¹, Kelly Giudici¹, Philipe De Souto Barreto¹, Bruno Vellas¹ (1. Gerontopole Toulouse — Toulouse (France))

Background: Apelin and GDF-15 have been proposed as biomarkers of age-related sarcopenia but evidence in human models is scarce. This study aimed to explore the associations between blood apelin and GDF-15 with sarcopenia incidence and the evolution of sarcopenia components over two years in older adults >70 years. Methods: Subjects from the Multidomain Alzheimer Preventive Trial (MAPT) were included. Serum Apelin (pg/mL) and plasma GDF-15 (pg/mL) were measured. Outcomes included sarcopenia defined by the European Working Group on Sarcopenia in Older People (EWGSOP) and its determinants (appendicular lean mass [ALM] evaluated through a Dual-energy X-ray Absorptiometry (DXA) scan, handgrip strength (HGS) and the 4-meter gait speed) measured over 2 years. Linear mixed models and logistic regression were used to explore the longitudinal associations. Results: We included 168 subjects from MAPT (median age=76y, IQR=73–79; 78% women). Serum apelin was not significantly associated with sarcopenia incidence (OR=1.001;95%CI=1.000,1.001;p-value>0.05 in full-adjusted models) nor with ALM (β=-5.8E-05;95%CI=-1.0E-04,2.12E-04;p>0.05), HGS (β=-1.1E-04;95%CI=-5.0E-04,2.8E-04;p>0.05), and GS (β=-5.1E-06;95%CI=-1.0E-05,2.0E-05;p>0.05) in fully adjusted models. Similarly, plasma GDF-15 was not associated with both the incidence of sarcopenia (OR=1.001,95%CI=1.000,1.002,p>0.05) and the evolution of its determinants ([ALM, β=2.1E-05;95%CI=-2.6E-04,3.03E-04;p>0.05], HGS [β=-5.9E-04;95%CI=-1.26E-03,8.1E-05; p>0.05] nor GS [β=-2.6E-06;95%CI=-3.0E-05, 2.3E-05;p>0.05]) in fully adjusted models. Conclusions: Blood apelin and GDF-15 were not associated with sarcopenia incidence or with the evolution of sarcopenia components over a 2-year follow-up in community-dwelling older adults. Well-powered longitudinal studies are needed to confirm or refute our findings. Conflict of interest: None

LP24 — CYB5R3 Overexpression Modulates Inflammation, Body Weight and Body Composition in Female Mice Fed Long-Term a Nicotinamide Riboside-Supplemented Diet

Luz Marina Sánchez-Mendoza¹, Carlos Pérez-Sánchez², Isabel M. Burón¹, José Antonio González-Reyes¹, Chary López-Pedrera², Rafael De Cabo³, José Manuel Villalba⁴ (1. Department Of Cell Biology, Physiology And Immunology, Faculty Of Sciences, University Of Córdoba, Agrifood Campus Of International Excellence (ceia3). — Córdoba (Spain), 2. Imibic, Rheumatology Service, Imibic/reina Sofía Hospital/university Of Córdoba — Córdoba (Spain), 3. Translational Gerontology Branch, National Institute On Aging, National Institutes Of Health — Baltimore (United States), 4. Department Of Cell Biology, Physiology And Immunology, Faculty Of Sciences, University Of Córdoba, Agrifood Campus Of International Excellence (ceia3). — Córdoba (United States))

Background: Previous studies developed in our group have allowed to characterize NADH-cytochrome b5 reductase-3 (CYB5R3) as a new pro-longevity gene. These studies have documented that CYB5R3 contributes to maintain respiratory metabolism, protects against oxidative stress, and prevents cellular senescence in transgenic mice overexpressing CYB5R3 (TG mice). Since NAD+ levels decrease with aging, therapies aimed at preserving this molecule are of extreme interest as potentially anti-aging interventions. Several metabolites of the NAD+ biosynthesis salvage pathway, as nicotinamide riboside (NR), act as NAD+ boosters that have been shown in most cases to improve metabolic health with aging. Existing data on the different susceptibility to diseases and the effect of drugs in males and females demonstrate the existence of sexual dimorphism, highlighting the importance of including both males and females in biomedical studies. However, most studies involving NAD+ boosters have been conduced in males, and there is also evidence supporting that high NAD+ levels could promote a proinflammatory phenotype and deteriorate metabolic health, particularly in the females. Objectives: Our aim was to study the long-term effects of NR supplementation in female mice overexpressing or not CYB5R3, in terms of body weight, body composition and inflammation. Methods: Studies were carried out with female wild-type and CYB5R3-TG mice in a C57BL/6 background, that were fed for 4 (adult) or 21 months (old) an AIN93M diet, either supplemented or not with NR (NIAGEN®, kindly supplied by ChromaDex Inc., Irvine, CA, USA) at a daily dose of 400 mg per kg body weight, starting at the age of 3 months. CYB5R3 overexpression in TG mice was confirmed by Western blot. Body weight data were recorded weekly throughout the dietary intervention. Determinations of body composition were carried by NMR using an EchoMRI™ equipment. C Reactive Protein (CRP) values were determined at the end of each nutritional intervention with blood samples obtained from mice that had been fasted since the day before sacrifice. Body weight increased with aging in NR-supplemented wild-type mice, but this effect was prevented by CYB5R3 overexpression. Interestingly, CYB5R3 overexpression abolished the accumulation of fat promoted by NR both in adult and old females. Conversely, lean mass and lean-to-fat ratio were decreased in NR-supplemented wild-type mice, which was reverted by CYB5R3 overexpression. Measurement of C reactive protein (CRP), as overall marker of proinflammatory state, revealed a proinflammatory effect of NR in adult females, which was completely abated by CYB5R3 overexpression. However, NR supplementation decreased CRP levels regardless the CYB5R3 status in old females. Conclusion: In the long term, NR affects female mice by increasing body weight and the amount of body fat, and increases inflammation in adult females, but these effects can be overcome by CYB5R3 overexpression. In conclusion, CYB5R3 overexpression might maximize the beneficial outcomes of an intervention based on NR supplementation along aging by preventing potentially deleterious side-effects that have been associated to NAD+-boosting therapies in the females. We are currently developing further studies to elucidate the molecular mechanisms underlying our findings.

LP25 — A Narrative Review of Mitochondrial Dysfunction and Intrinsic Capacity

Jaqueline Aragoni Da Silva¹, Laurent Martinez², Yves Rolland¹, Bruno Vellas¹, Philipe Souto Barreto¹ (1. Institute Of Aging, Gérontopôle Of Toulouse, Centre Hospitalier Universitaire De Toulouse — Toulouse (France), 2. Institute Of Metabolic And Cardiovascular Diseases, I2mc, Inserm, Umr 1048 — Toulouse (France))

Background: Based on a life course approach, a new concept of healthy aging has been proposed, considered as the achievement of wellbeing through optimizing functional ability, and incorporates the interaction of environment and intrinsic capacity (IC). IC is a holistic, person-centered and integrated approach that includes five domains, namely locomotion, psychological, cognition, vitality, and sensory. IC can potentially be a tool for screening function over lifespan. Mitochondrial dysfunction is considered a hallmark of aging. Age-related declines on mitochondria function include decreasing mitochondrial enzymes, mitochondrial respiratory capacity, phosphocreatine recovery time, and increasing reactive oxygen species. Objectives: to review the scientific evidence conducted in humans on the association of mitochondrial dysfunction and IC domains. Methods: a literature search was conducted in October 2021 on PubMed, inserting terms related with mitochondria, aging and each IC domains. It included studies in English conducted in humans in the context of aging that investigated the association of mitochondrial dysfunction with IC domains. IC domains were considered in the perspective of Integrated Care for Older People (ICOPE), according to World Health Organization guidance for assessment of: Locomotion. Short Physical Performance Battery (SPPB) and its components; Psychological. Validated scales (Geriatric Depression Scale (GDS), Patient Health Questionnaire (PHQ-9), etc.); Cognition. Validated scales (Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), etc.); Vitality (Handgrip strength); Hearing loss. Audiometry, validate scales and performance tests (Whispered Voice Test); Vision impairment. Validated scales and performance tests (Snellen Chart). Post-mortem studies of age-related hearing and vision impairment were also considered. Results: Locomotion: mitochondrial dysfunction has been related to performance-based tests of mobility and that includes not only muscle skeletal mitochondrial function but also blood-measured mitochondrial function. Cross-sectional studies showed higher skeletal muscle mitochondrial efficiency and oxidative capacity were associated with greater gait speed. Enrichment of peripheral blood mononuclear cells (PBMCs) mitochondrial ATP production was related to improvement in gait speed. Mitochondrial protein expression and contents has also been linked to gait speed, including proliferator-activated receptor c coactivator a (PGC-1a), skeletal muscle mitochondrial apoptotic signaling proteins and acylcarnitine levels. Low-functioning older adults, compared to high-functioning, displayed higher age-related mitochondrial dysfunction, impaired iron regulation, markers of mitochondrial autophagy, and decreased levels of oxygen consumption. Psychological: cross-sectional findings showed that levels of mitochondrial DNA (mtDNA) copy number were different between depressed and non-depressed individuals, revealing that mitochondrial dysfunction appears to play a role in depression. However, longitudinal investigations did not identify a clear pattern on these changes. Yet, the mechanisms underlying this relationship are not elucidated and may be affected by aspects related to the level of disease severity, the body tissue investigated and the duration of the disease. Cognition: studies indicate mitochondrial dysfunction could be one mechanism of cognitive impairment, showing mitochondrial content in brain-areas and in other body fluids seems to be distinct between normal and impaired cognitive older adults. Lower levels of peripheral leukocyte mtDNA copy number, and decreased mitochondrial bioenergetics in fibroblasts from peripheral cells were identified in older adults with cognitive impairment and sporadic Alzheimer’s Disease, respectively, compared to healthy older adults. In addition, increases on PBMCs mitochondrial ATP production and electron flux were related to improvement in cognition function. Vitality: greater PBMCs mitochondrial spare respiratory capacity and maximal respiration were cross-sectionally correlated with better handgrip strength. Elements related to mitochondrial function, such as mitokines and acylcarnitine, have been proposed to be related to handgrip strength. However, whether the beneficial or detrimental role that these mitochondrial dysfunction-related contents play on handgrip strength is not clear and may vary according to the level of stress and interactions with other factors, such as inflammation. Sensory: among individuals with age-related macular degeneration (AMD), retinal pigment epithelium had a distinct pattern of age-related mitochondrial deterioration, presenting greater and faster decrease in amount and size of mitochondria, compared to normal aging. AMD individuals also showed higher mtDNA damage and alterations in proteins related to mitochondrial translation, import of proteins encoded in the nuclei and related to metabolism. In addition, the greater the mtDNA damage, the greater the AMD severity. Individuals with age-related hearing loss had lower levels of peripheral blood mtDNA copy number and accumulation of certain levels of multiple mtDNA deletions in the cochlea tissue is suggested to be one of the mechanisms explaining age-related hearing loss. Conclusion: evidence supports the notion that mitochondrial dysfunction and its related-contents may play a role on IC, and that may occur by distinct pathways between IC domains. Although most evidence comes from cross-sectional and small sample sizes, with distinct population characteristics, contexts and methodological assessments, several lines of evidence support the notion that mitochondrial function is a potential target for improving IC domains. Further investigations about mitochondrial function and IC will help to elucidate the specific mechanisms by how amelioration of mitochondrial function can contribute to a better healthy aging. The authors declare no conflict of interest.

LP26 — White Adipose Tissue-Muscle Cross Talk During Sarcopenia: The Crucial Role of Haptoglobin in Regenerative Processes Alteration

Marie Catenacci¹, Ophelie Pereira¹, Lucas Bourdens¹, Philippe Valet¹, Jean-Philippe Pradère¹, Cédric Dray¹ (1. Institut RESTORE, UMR 1301, Institut National de la Santé et de la Recherche Médicale (INSERM), -CNRS-Université Paul Sabatier, Université de Toulouse — Toulouse (France))

In 2015, the WHO recommended to keep five intrinsic capacities in order to maintain an healthy aging: cognition, psychosocial, senses, vitality and locomotion. Locomotion is directly linked to an age-related pathology: the sarcopenia. The prevalence of sarcopenia is reported to affect 40% in individuals older than 60 years. Even with a conservative estimate of prevalence, sarcopenia affects more than 50 million people today and will affect 200 million individuals in the next 40 years. This disease is described as an aged-associated progressive loss of muscle mass and strength over the time. It is defined by a protein synthesis/proteolysis imbalance, an alteration of mitochondria-related metabolism together with a higher oxygen species release, and a proinflammatory status in myocytes. Moreover, within the skeletal muscle, the loss of regenerative capacities participates to the onset of sarcopenia. In this context, new strategies are needed to slow down the different processes of sarcopenia. To do so, our working hypothesis is based on interorgan communication and more specifically on adipocyte-muscle crosstalk during aging. Indeed, during aging, adipose tissue secretions are deeply modified toward a proinflammatory phenotype especially in visceral adipose tissue. This change could participate to the acceleration of age-related pathologies onset such as sarcopenia and more specifically target muscle regeneration. Among these changes, we previously described that haptoglobin secretion by visceral adipose tissue is increased with age. Haptoglobin is a glycoprotein involved in acute phase of inflammation and had an important antioxidant role by carrying hemoglobin. Interestingly, previous data reported that haptoglobin deficient mice (Hp-/-) exhibited muscle atrophy related to an increase of oxidative stress. Moreover, haptoglobin receptors (CCR2 and CD163) are two receptors found on the surface of immune cells (monocyte and macrophages) and have important role in muscle regeneration processes. Objectives: The main objective is to study the role of haptoglobin in the adipocyte-muscle crosstalk during age-associated sarcopenia and muscle regeneration. To do so, we focused our attention on haptoglobin role during cardiotoxin-induced regeneration by studying myogenic and immune systems. Methods: To assess the effects of haptoglobin during regenerative processes in vivo, we promoted muscular regeneration through unilateral intramuscular injections of cardiotoxin in tibialis and gastrocnemius of 3 or 12-months-old mice. Contralateral muscle are injected with PBS as control. In parallel, recombinant haptoglobin was daily i.p injected in order to mimic the increase of plasma haptoglobin observed in aged and sarcopenic mice. During the protocol, strength was measured by a functional grip test to evaluate the muscle functional capacity after regeneration. Then, blood and skeletal muscles (gastrocnemius) were collected 2, 7 and 14 days after cardiotoxin injection. Plasma haptoglobin level has been determined by ELISA and histology, transcriptomic and cell population analyses (flow cytometry) were performed in both injured and control muscles in order to evaluate the effect of haptoglobin. In vitro, we determined the ability of haptoglobin to control muscle cells proliferation in C2C12 cells. Results: In 3-months-old mice, 14 days of haptoglobin treatment induces a decrease of functional muscle abilities (muscle strength), accompanied by a lower cross-sectional area in gastrocnemius. Moreover, cross-sectional area of nuclei-positive fibers is also reduced, reflecting a delay in regeneration. Consistently, we observed a delay in expression of genes involved in inflammatory and myogenic which are processes necessary for regeneration (Pax7, Myogenin, MyHC3, CD45, TNFa,…). Interestingly, the same results were observed in 12-months-old mice. To better understand the mechanisms involved, we analyzed potential cells targeted by haptoglobin treatment during muscle regeneration. For that, using flow cytometry, we studied the percentage of population of immune cells (neutrophils, monocytes, macrophages M1 and macrophages M2). With the treatment, the same percentage of CD45+ cells are observed but macrophages population is more important with haptoglobin treatment. Moreover, a preliminary result on culture of mouse muscular cells (C2C12) suggests a direct role of haptoglobin of myogenic cells. Indeed, haptoglobin treatment induce a lower number of cells after 4 days of proliferation, together with an increase of p38-MAPK phosphorylation. Next step will consist in studying the impact of haptoglobin on myogenic markers (MyHC3, eMyHC8, Pax7…), on signaling pathways and the percentage of myogenic cell populations (satellites cells, MyoD+ or Myogenin+ cells). Conclusion: Taken together, our data indicate that haptoglobin could be considered as future a therapeutic target to slow down the onset and the process of age-related sarcopenia. By acting on adipose tissue thought different aspect (nutrition, behavioral, pharmacology,…) the ultimate aim is to regulate the overproduction of haptoglobin associated with age and consequently reduced the incidence of sarcopenia and dependency.

LP27 — Anti-Aging Interventions With NAD+ Boosters and CYB5R3 Overexpression Induce Sex Dependent Mitochondrial Changes in Mice Kidney

Miguel Pérez-Rodríguez¹, Luz Marina Sánchez-Mendoza¹, Isabel María Burón¹, José Antonio González-Reyes¹, José Manuel Villalba¹ (1. Department Of Cell Biology, Physiology And Immunology, Faculty Of Sciences, University Of Córdoba, Agrifood Campus Of International Excellence (ceia3), Córdoba, Spain. — Cordoba (Spain))

Background: Aging is defined as the time-dependent gradual loss of organism functionality. Interventions aimed at maintaining or increasing NAD+ levels have engaged a great interest since the maintenance of this compound has been related with the prevention of metabolic diseases and the promotion of healthy aging. Among them, cytochrome b5 reductase 3 (CYB5R3) overexpression and dietary supplementation with NAD+ boosters have reported relevant results, being considering as anti-aging interventions. In the aging-related field, mitochondria have a leading role. It has been appreciated that aging is accompanied by an alteration of mitochondrial status, that could be related with a reduction of mitochondrial dynamics, a reduction of mitochondria turnover and the emergence of larger and aberrant mitochondria. Objectives: The aim of our studies was to analyze in kidney from male and female mice how CYB5R3 overexpression and/or dietary supplementation with the NAD+ booster nicotinamide riboside (NR) modify key markers of mitochondrial function, in order to understand how these interventions might affect old mice and to define a basis for future aging studies. Methods: Mitochondrial biomarkers, as well as levels of CYB5R3, were assessed by Western blot and the software Image Lab. The planimetric analysis of mitochondria from convoluted proximal tubule epithelial cells was performed using transmission electron microscopy images and the open source software ImageJ. Kidney samples were obtained from male and female 7-month-old adult mice of wild-type and CYB5R3-transgenic genotypes. Animals were fed ad libitum with a standard chow from weaning until they reached 3 months of age. Thereafter, they were transferred to a purified AIN93M diet, supplemented or not with NR (NIAGEN®, kindly supplied by ChromaDex Inc., Irvine, CA, USA) at a daily dose of 400 mg per kg body weight until they were euthanized. Results: The levels of CYB5R3, VDAC (mitochondrial mass marker), PGC-1alpha (master regulator of mitochondrial biogenesis), Fis1 (mitochondrial fission marker) and PARKIN (mitophagy marker) — through Western blot — as well as mitochondrial area (mitochondrial size marker) and mitochondrial circularity (mitochondrial shape marker) — through transmission electron microscopy — were evaluated as a function of sex, genotype, and diet. Results depicted a consistent pattern of changes. On the one hand, Western blot results indicated that CYB5R3 overexpression induced the upregulation of the biomarkers in both sexes. In wild-type mice, NR supplementation produced similar effects to those of CYB5R3 overexpression, and this effect was also observed for both sexes. However, the effect of NR supplementation in CYB5R3-transgenic mice was found dependent of sex, increasing the levels of the biomarkers in males while decreasing in females. On the other hand, electron microscopy results showed that CYB5R3 overexpression decreases the mitochondrial area in male mice, while producing no change in the females. NR dietary supplementation reduced the mitochondrial area in females of both genotypes, while did not alter mitochondrial area in wild-type males and decreased the area in transgenic males. Regarding mitochondrial circularity, CYB5R3 overexpression did not affect the mitochondrial area in males, while increased the size in females. NR supplementation did not alter mitochondrial circularity in wild-type males but increased this parameter in wild-type females, and decreased circularity in transgenic mice from both sexes. Conclusion: NR supplementation produces a pattern of changes in the analyzed kidney markers which is similar to that produced by CYB5R3 overexpression, both in males and females, while NR supplementation enhances the effects of CYB5R3 overexpression in males, but reverts the effects in females. Those effects produced by NR supplementation in kidney from wild-type mice of both sexes can be interpreted as beneficial in relation with the hallmarks of aging. In CYB5R3 transgenic mice however, NR supplementation seems to induce a sex dependent effect in the transgenics, with potentially detrimental effects in females, and potentially beneficial effects in males. The reported changes could determine how aging will affect these individuals as they get old.

LP28 — CYB5R3 Overexpression Maintains Mitochondrial Function and Autophagic Signaling in Skeletal Muscle of Aged Transgenic Mice

Sara López-Bellón¹, Sandra Rodríguez-López¹, Rafael De Cabo², Isabel Burón¹, José Antonio González-Reyes¹, José Manuel Villalba¹ (1. Department Of Cell Biology, Physiology And Immunology, Faculty Of Sciences, University Of Córdoba, Agrifood Campus Of International Excellence (ceia3). — Córdoba (Spain), 2. Translational Gerontology Branch, National Institute On Aging, National Institutes Of Health, Baltimore, Md, USA. — Baltimore (United States))

Background: Aging is defined as a gradual decline of the normal physiological functions of an organism, and is considered as the most important risk factor for most chronic diseases. The maintenance of an optimized mitochondrial function and the capacity to remove damaged structures by autophagy are two of the main hallmarks that define successful aging. Nowadays, there is great interest in elucidating the mechanism of aging to create therapies to delay aging. Some of these therapies are caloric restriction, physical exercise, and the upregulation of enzymes which promote healthy aging and increase longevity. Cytochrome b5 reductase 3 (CYB5R3) catalyzes electron transfer from NADH to cytochrome b5 and also to alternative electron acceptors as plasma membrane coenzyme Q or several exogenous compounds. CYB5R3 overexpression activates respiratory metabolism in model organisms, and has a prolongevity effect in transgenic mice, mimicking some of the salutary effects of calorie restriction. It is however unknown how CYB5R3 overexpression targets key pathways that modulate the rate of aging in skeletal muscle, the postmitotic tissue with a greater contribution to resting energy expenditure. Objectives: The objective of this study is elucidating how CYB5R3 overexpression extends longevity. To achieve this objective, we have focused our efforts towards the study of mitochondrial metabolism, morphology and autophagy in hindlimb skeletal muscle, as a model of a postmitotic tissue which plays a relevant role in aging, from young-adult and old mice of wild-type (WT) and CYB5R3-overexpressing (TG) genotypes. Methods: Biochemical markers related with the function of mitochondria (electron transport complexes, fission and fusion dynamics proteins, and mitochondrial biogenesis factors), and key biochemical markers of general autophagy and mitophagy pathways were evaluated by western blot in hind limb skeletal muscle obtained from young-adult (7 months old) and old (24 months old) male mice of WT and TG genotypes. Coenzyme Q levels were measured by HPLC in lipids extracts. Ultrastructural features of subsarcolemmal and intermyofibrillar mitochondria were studied by electron microscopy in red gastrocnemius samples. Results: CYB5R3 was highly overexpressed in skeletal muscle and this protein was efficiently targeted to the mitochondria. Aging did not affect CYB5R3 levels in TG, but produced an increase in WT mice. Old TG mice exhibited increases of complexes I, II and IV. Moreover, CYB5R3 overexpression prevented the aging-related decrease of complexes I, III, IV and V. Aging produced a substantial decrease of MFN-2 in WT mice but not in TG, reinforcing the importance of CYB5R3 expression to prevent aging-associated mitochondrial dysfunction in skeletal muscle. Both aging and CYB5R3 overexpression upregulated markers of mitochondrial fission (FIS1, MFF and DRP-1), although with different outcomes on mitochondrial ultrastructural features: aged WT mice exhibited mitochondrial fragmentation whereas CYB5R3 overexpression increased mitochondrial size in old TG mice concomitant with an improvement of autophagic recycling. The decrease produced by aging in the abundance of mitochondrial biogenesis markers TFAM and NRF-1 and in the levels of coenzyme Q was not prevented by CYB5R3 overexpression. Conclusions: Skeletal muscle is a suitable model to study the direct effects of CYB5R3 overexpression in the cellular physiology. Overexpression of CYB5R3 in TG mice effectively counteracts mitochondrial dysfunction and the decline in autophagy that is associated with aging in skeletal muscle. Interventions that stimulate CYB5R3 activity could represent a valuable strategy to counteract the deleterious effects of aging in this tissue.

LP29 — Mitochondria Heterogeneity in Skeletal Muscle During Zebrafish Ageing

Eléna Morin¹, Marie-Laure Renoud¹, Marie Sallese¹, Bruno Payre², Romain Madelaine³, Cédric Dray¹, Aurélie Quillien¹, Jean-Philippe Pradère¹ (1. Institut RESTORE, UMR 1301, Institut National de la Santé et de la Recherche Médicale (INSERM), -CNRS-Université Paul Sabatier, Université de Toulouse — Toulouse (France), 2. Centre de Microscopie Electronique Appliquée à la Biologie, Faculté de Médecine de Rangueil, Université de Toulouse -Toulouse (France), 3. Davis Center for Regenerative Biology and Medicine, MDI Biological Laboratory — Bar Harbor, ME (United States))

Background: Societies of developed and under-developed countries are adapting to population aging due to significant increase in life expectancy, which represents now a major challenge for health care systems all over the world. To alleviate the economic burden, there is a need to decrease dependency of aging population and promote healthy aging. Frailty syndrome is a clinical consequence of age-associated functional decline which clinically translates in muscle sarcopenia. Sarcopenia is a progressive and systemic skeletal muscle disorder involving the accelerated loss of muscle mass and function, associated with increased adverse outcomes including falls, functional decline, frailty, and mortality. Several molecular mechanisms have been described as potential causes for the etiology of sarcopenia. The mechanisms notably related to hormone function (e. g. IGF-1 and Insulin), muscle fiber composition and neuromuscular drive, myo-satellite cell proliferation and differentiation, or dysregulation of proteostasis have been proposed to play a crucial role but recently mitochondria functions has drawn a lot of attention in the field. Mitochondria are affected by ageing (and vice-versa) to very different extents, depending on the tissue and the species. Mitochondria are heterogeneous and highly dynamic organelles and their functions are subjected to complex regulations through modulation of its biogenesis, bioenergetics, dynamics and clearance (mitophagy) within cells. Mitochondria dysfunctions have been previously associated with sarcopenia progression exemplified by defect in mitophagy, increased ROS production and heteroplasmy. Mitochondria are thoroughly characterized regarding their function in both energy production (ATP synthesis) and regulation of cell death; however, some other features of mitochondrial biology have been poorly depicted. Among them, the heterogeneity that exists within mitochondria populations is not really understood. Objectives: In this context, we aim to study mitochondria heterogeneity in several tissues clinically related to aging especially blood and skeletal muscles. We will also investigate whether mitochondria size heterogeneity is related to ultrastructural heterogeneity and associated with mitochondria dysfunction related to sarcopenia. In addition, functional analysis will be performed to firmly establish if mitochondria size heterogeneity is related to a functional heterogeneity. We will also try to demonstrate in vitro and in vivo that certain mitochondrial subpopulation may play a detrimental role in muscle physiology while others may exert a protective role. Methods: To address this question, we took advantage of several fish models such as zebrafish, TERT KO zebra fish, and the Turquoise African killifish. On each fish line, the isolated mitochondria were stained using different fluorescent probes (MitoTracker, TMRM or MitoSox) and size heterogeneity was evaluated using Flow cytometer and eventually sorted out using a FACS Aria cytometer. Electronic microscopy approaches combined with deep learning assisted-image analysis were also used to confirm data generated by the flow cytometry. Functional analysis combining bioenergetics, lipidomics, proteomics and mtDNA sequencing were performed on each mitochondria sub-population. We finally functionally tested in vitro and in vivo the functional capacity of each mitochondria subpopulation by using mitochondria transfert, notably by using a zebrafish fluorescent reporter line where specific mito-TOM20 protein was fused with a red fluorescent protein. Results: We report a very high heterogeneity in mitochondria size, structure, and functional properties in skeletal muscle already in a young individual. Moreover, this heterogeneity is aggravated with ageing and sarcopenia, notably with a shift toward bigger mitochondria. We identified pro and anti-aging mitochondrial subpopulations during sarcopenia progression in terms of size and structure (cristae), which may also differ in ROS production and bioenergetic flexibility. Conclusion: A better characterization of mitochondria heterogeneity at different physiological level in sarcopenia development will help to identify new mechanisms underlying muscle loss progression. Using this multiscale approach, we hope to elucidate the complexity of mitochondria biology and identify new targets that might be helpful to slow down mitochondria dysfunctions during ageing.